Microfluidic assay assemblies and methods of manufacture

ABSTRACT

In assembling a portion of a microfluidic device by conducting bonding action by contacting faces of opposed bondable materials, one comprising a flexible sheet, the method, while maintaining continual contact of the faces in a region R2 until bonding is completed, of employing repeated make-and-break-contact manufacturing protocol on a region R1 of the contacted faces of the bondable materials, thereby over time neutralizing the tendency for permanent bonds to form in the region R1, thus to enable making and breaking actuated movements of the region R1 of the flexible sheet relative to the portion of the other material that it opposes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation in Part of International ApplicationNo. PCT/US2013/000056, filed Mar. 8, 2013, which claims priority toProvisional Application No. 61/608,570, filed Mar. 8, 2012, ProvisionalApplication No. 61/754,377, filed Jan. 18, 2013, and is a Continuationof U.S. application Ser. No. 13/427,857, filed Mar. 22, 2012, now U.S.Pat. No. 9,216,412 B2, which claims priority to U.S. ProvisionalApplication No. 61/465,688, filed Mar. 22, 2011 and U.S. ProvisionalApplication 61/608,570, filed Mar. 8, 2012, and U.S. application Ser.No. 13/427,857 is a Continuation in Part of International ApplicationNo. PCT/US10/57860, filed Nov. 23, 2010, which claims priority to U.S.Provisional Application No. 61/263,572, filed Nov. 23, 2009, and U.S.application Ser. No. 13/427,857 is also a Continuation in Part ofInternational Application No. PCT/US11/29736, filed Mar. 24, 2011, whichis a Continuation in Part of International Application No.PCT/US10/57860, filed Nov. 23, 2010, which claims priority to U.S.Provisional Application No. 61/263,572, filed Nov. 23, 2009; and thisapplication is a Continuation in Part of International Application No.PCT/US2013/033610, filed Mar. 22, 2013, which claims priority toProvisional Application No. 61/754,377, filed Jan. 18, 2013, and is acontinuation of U.S. application Ser. No. 13/427,857, filed Mar. 22,2012, now U.S. Pat. No. 9,216,412 B2, which claims priority to U.S.Provisional Application No. 61/465,688, filed Mar. 22, 2011 and U.S.Provisional Application 61/608,570, filed Mar. 8, 2012, and U.S.application Ser. No. 13/427,857 is a Continuation in Part ofInternational Application No. PCT/US10/57860, filed Nov. 23, 2010, whichclaims priority to U.S. Provisional Application No. 61/263,572, filedNov. 23, 2009, and U.S. application Ser. No. 13/427,857 is also aContinuation in Part of International Application No. PCT/US11/29736,filed Mar. 24, 2011, which is a Continuation in Part of InternationalApplication No. PCT/US10/57860, filed Nov. 23, 2010, which claimspriority to U.S. Provisional Application No. 61/263,572, filed Nov. 23,2009. This application hereby incorporates by reference, in itsentirety, each and every application referred to above, to the extent ofmatter related to any of the features defined in the “Summary” sectionof this application. This application is also a Continuation in Part ofU.S. application Ser. No. 13/427,857, filed Mar. 22, 2012, now U.S. Pat.No. 9,216,412 B2, which claims priority to U.S. Provisional ApplicationNo. 61/465,688, filed Mar. 22, 2011 and U.S. Provisional Application61/608,570, filed Mar. 8, 2012, and U.S. application Ser. No. 13/427,857is a Continuation in Part of International Application No.PCT/US10/57860, filed Nov. 23, 2010, which claims priority to U.S.Provisional Application No. 61/263,572, filed Nov. 23, 2009, and U.S.application Ser. No. 13/427,857 is also a Continuation in Part ofInternational Application No. PCT/US11/29736, filed Mar. 24, 2011, whichis a Continuation in Part of International Application No.PCT/US10/57860, filed Nov. 23, 2010, which claims priority to U.S.Provisional Application No. 61/263,572, filed Nov. 23, 2009; and thisapplication is a Continuation in Part of U.S. application Ser. No.13/511,593, filed Sep. 24, 2012, now U.S. Pat. No. 9,279,001 B2, whichclaims priority to International Application No. PCT/US2010/057860,filed Nov. 23, 2010, which claims priority to U.S. ProvisionalApplication No. 61/263,572, filed Nov. 23, 2009. This application herebyincorporates by reference, in its entirety, U.S. application Ser. No.13/427,857, filed Mar. 22, 2012 to the extent of matter related to anyof the features defined in the “Summary” Section of this application.

TECHNICAL FIELD

The invention concerns assays in microfluidic systems, including systemsthat employ portable microfluidic devices. Some versions of microfluidicdevices are in the form of microfluidic cartridges (cassettes) that areactuated and read by an associated apparatus such as a bench-topinstrument that both conducts the assay protocol within the cartridgeand reads the results.

The invention also concerns multiplex microfluidic assays in whichmultiple assays performed in a microfluidic system are read or scannedwith epi-fluorescence.

The invention in particular relates to monitoring assays performedwithin microfluidic systems, to detecting assay results aftermicrofluidic assays have been run (performed), and to determining theprecise relative location of a microfluidic system to a precisedetection system, for conducting monitoring or detection with precision.

The invention has broad aspects that are applicable to microfluidicassay systems, in general, and more specific aspects that concern theassays conducted within portable microfluidic cartridges, andparticularly cartridges in which the relative position of the cartridgeand a precise outside scanning system is not precisely determined.Particularly important applications of the invention concernmicrofluidic assay cartridges that are inserted into a multi-functionapparatus that both causes the assay to be performed within thecartridge and the results detected.

BACKGROUND

As is well understood, with any microfluidic assay system there is thepotential for failure, and with complex systems, typically, there arenumerous potential failure modes. Examples of failure modes formicrofluidic assay systems relate to flows in microfluidic channels andto valves and pistons that control the flows according to apre-determined assay protocol. The failure modes occur with anymicrofluidic system, but can be of particular concern when the assay isperformed within a microfluidic cartridge. Blockage of a microfluidicchannel and inability of a valve to open or close are examples offailure. If a valve does not open, flow is prevented; if it does notclose completely, valve leakage may occur at an inopportune time. Thereis also the possibility of a contaminant in the microfluidic channels.

There are also potential for human errors. For example, most samples forimmunoassays, e.g., human plasma or human serum samples, are dilutedwith a diluent at a prescribed ratio, for example one-to-one (one partsample to one part diluent) or one to five. It is important for theproper ratio to be supplied, but personnel may improperly prepare thesamples.

With microfluidic assays in general, and especially automatedmicrofluidic immunoassays performed within portable cartridges, thereare many steps in the assay protocol that need to occur with specifictiming and specific reagents. For instance it is necessary to know whena buffer liquid or reagent is being flowed through a microfluidicchannel and when a sample is being flowed, and whether, in each case, itis flowed at the proper rate and/or for the proper duration. It is alsonecessary to know whether there is leakage or flow into regions where noflow can be permitted. Further, when a liquid volume is displaced in apulsed flow type microfluidic system, for instance as a reciprocatingpiston pump pushes small slug (portions) of liquid sequentially througha channel, it is important to have a precisely determined quantity offluid in each slug.

Problems in attempting to obtain this information arise. For instance,because liquids flow in microfluidic channels are very tiny (e.g.,100-200 microns cross section width and depth, only millimeters inlength) flows are difficult to visualize. The channels are so small thatit is difficult for the human eye to observe the fact that liquid is notflowing where it is desired. The assay reagents are typicallytransparent, compounding the difficulty of visual or opticalobservation.

The problems are especially acute when seeking highly accuratequantification in a microfluidic assay. In quantifying assays it isdesired that a given amount of immobilized capture agent be exposed to agiven amount of various fluids to enable reactions over defined times sothat results can be compared to a standard to enable the quantification.Results need to be determined with an overall coefficient of variationof less than 10% (accuracy within 10%), preferably much less.

Thus, to be quantitative, assays require consistent run-to-runperformance. For example in an assay employing a fluorescent dyeconjugated with immobilized, captured moieties, the concentration andthe volume of the buffer or wash liquid, of secondary reagents such asantibodies, and of fluorescent dye all need to be the same from run torun if one is to compare the result to a standard calibrating curveprecisely generated from previous calibrating runs. This is particularlytrue in blood testing in which a patient human plasma or serum sample ismeasured for the presences or the quantity of specific health-relatedanalytes, for instance, antibodies such as interleukins (a class ofantibodies called cytokines), e.g., IL5 or IL6. There are many otherclasses of antibodies to be measured in plasma or serum.

For these reasons it is important to verify that at the end of an assaywhen a result is generated, that the result has been produced preciselyaccording to the desired protocol.

The result of an assay is typically measured by detecting an emanation,e.g., a fluorescence intensity, from a reaction site. The emanation maycome from a bead, a micro particle or an immobilized spot. As presentlypreferred, it comes from an immobilized glass nano-reactor (GNR) in theform of a small hollow tube or micro-tube, of length no more than 1000micron, typically less than 500 micron, with capture agent, e.g.,antibody, immobilized on its inside surface.

The fluorescence intensity from the region of the capture agent isessentially all that is measured at the completion of many assays. Thatfluorescence intensity is compared to a calibration curve. From thecalibration curve the unknown concentration of the analyte isdetermined. For the calibration curve to be valid to a particular run,it is necessary that all of the conditions for that assay are repeatedspecifically and reproducibly from run to run. Improved means to measuresuch conditions are to be greatly desired.

For the following description of novel techniques for monitoringmicrofluidic assays, it is important that the exact location of featureson a microfluid cassette be known. Novel techniques for doing this aredescribed later herein.

SUMMARY

In a first aspect, the invention features in assembling a portion of amicrofluidic device by conducting bonding action by contacting faces ofopposed bondable materials, one comprising a flexible sheet, the method,while maintaining continual contact of the faces in a region R2 untilbonding is completed, of employing repeated make-and-break-contactmanufacturing protocol on a region R1 of the contacted faces of thebondable materials, thereby over time neutralizing the tendency forpermanent bonds to form in that region R1 thus to enable making andbreaking actuated movements of the region R1 of the flexible sheetrelative to the portion of the other material that it opposes.

Preferred implementations of this aspect of the invention mayincorporate one or more of the following:

The faces of opposed bondable materials may have the capability offorming molecular bonds, and molecular bonds between the faces may beformed in the region R2 of the continual contact. The materials may havethe capability of forming covalent bonds and covalent bonds between thefaces may be formed in the region R2 of the continual contact. At leastone of the materials may be a surface-activated bondable material. Atleast one of the materials may be surface-activated PDMS. Both materialsmay be PDMS. The flexible membrane portion may define a valve diaphragmand the opposed portion of the other material may define a valve seatwhich the flexible membrane portion engages. The bonding of the flexiblesheet material to the opposed material simultaneously may act tocomplete and seal a flow channel, may fix the position of an insertedelement in a flow channel, or may form the diaphragm of the piston of amembrane pump. All actions may be simultaneously performed. The repeatedmake- and break-contact manufacturing protocol may be performed byapplying, respectively, positive and negative air pressure to the backof the flexible membrane portion. The negative air pressure may be oflarger magnitude than the positive air pressure. The method may includeproviding a chamber defining a deflection cavity into which the flexiblemembrane portion may be drawn away from contact with the oppositematerial while other regions of the faces of the two materials are incontact for bonding; bringing the regions of the faces of the twomaterials into continuing contact for bonding and applying vacuum to thedeflection cavity to draw the flexible membrane portion into the cavity,away from the opposite material; thereafter, in cyclical manner,repeatedly applying a positive pressure pulse followed by a negativepressure pulse to cause the flexible membrane portion to repeatedly makeand break contact with a surface portion of the opposite material duringcure of the bond between adjacent surface portions of the two materialsthat are in continuing contact. The vacuum may be applied to thedeflection cavity prior to and during initial contacting of the regionsof the faces of the two materials for bonding, so that the membraneportion does not bond at the time of initial contact of the regions ofthe faces of the materials. The material may comprise the flexible sheethas its face comprised of PDMS, the face having been surface-activated.The material may be a layer comprising PDMS throughout its thickness.The flexible sheet may be comprised substantially of a material otherthan PDMS, but may have its face opposing the other material comprisedof an activatable adhesive coating that has been surface-activated forbonding. The activatable adhesive may be PDMS. The material opposite theflexible sheet may have its face for bonding comprised of PDMS, the facemay be surface-activated. The material opposite the flexible sheet mayhave its face for bonding comprised of glass, presenting OH groups tothe activated PDMS surface. The material opposite the flexible sheet mayhave a main body formed of a material, on the face of which may be alinker layer bonded to that material, which may present OH groups to theactivated surface of the PDMS membrane portion. The material oppositethe flexible sheet may comprise synthetic resin. The synthetic resin maybe one of the group comprising polymethacrylate, polystyrene, orpolysulfane, which has been surface-activated and bonded to the linkerlayer. The material opposite the membrane portion may not deflect duringthe bonding process. The material opposite the flexible sheet having aflexible membrane portion may also have a flexible sheet the flexiblemembrane portion opposite the first a flexible membrane portion;including, providing, at the face of the second membrane, a secondchamber defining a deflection cavity into which the second flexiblemembrane portion may be drawn away from contact with the oppositematerial while other regions of the faces of the two materials are incontact for bonding; bringing the faces of the two materials intocontinuing contact for bonding and applying vacuum to the deflectioncavities to draw each of the flexible membrane portions into itsrespective cavity, away from the opposite material; thereafter, incyclical manner, repeatedly, in synchronized manner, applying a positivepressure pulse followed by a vacuum pulse to each of the chambers tocause the membrane portions to repeatedly make and break contact withthe opposite membrane during cure of the bond between adjacent faceportions of the two materials that are in contact. The flexible membraneportion may be an elastomer subject to thinning under tension (asdescribed by Poisson), and the membrane portion may be deflectedinitially with a negative pressure of a first magnitude, and later at amagnitude of a second magnitude of substantially larger value, theinitial value assuring bonding of contacting may face up to theperimeter of the flexible membrane portion. A microfluidic structure maybe produced. The microfluidic structure may be in the form of apneumatically operable, hand held bio-assay cartridge containingmicrofluidic channels, reservoirs for sample and reagents and membranepumps and valves formed of flexible portions of the flexible sheet.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

The following descriptions of drawings are each respectively shown inaccordance with embodiments of the present invention.

DESCRIPTION OF DRAWINGS

FIG. 1 is an illustration of assembly steps for an assay cassette havingflow channels in which discrete micro-length tube flow elements (e.g.,GNRs) are fixed between a Fluidic Layer subassembly and a PDMS sheet ofa Control/Reservoir Layer subassembly (see also FIG. 27);

FIGS. 2A and 2B are diagrammatic plan views, on enlarged scale,depicting 4 micro-length tube elements fixed in series in a flowchannel, FIG. 2A illustrating substantial liquid flow both through theflow elements and as by-pass flow through by-pass passages on theoutside of the elements, while FIG. 2B illustrates the continued flowcondition in the case in which one micro-length tube becomes blocked,and FIG. 2C is a cross-section, on enlarged scale, depicting flowconditions in which a micro-length tube element becomes plugged (i.e.blocked);

FIG. 3 is a much enlarged perspective view of a portion of the cassette,denoting four parallel channels, in each of which are fixed sixmicro-length tube flow elements;

FIG. 4 is a flow diagram of steps A through K in the manufacture and useof the cassette (i.e. “flow chip”) constructed according to theforegoing Figures;

FIG. 5 depicts a flow element and a micro-length tube element example,illustrating the percentage reduction of active capture agent underdiffering conditions of the surfaces of the element;

FIG. 6 depicts a device employed to aggressively agitate a suspension ofmicro-length tube elements in a capture agent, e.g. antibody, antigen,or oligomer-containing liquid;

FIG. 7 illustrates diagrammatically, by vectors, sheer forces τ to whichthe outside and inside surfaces of the micro-tubular element areexposed;

FIG. 8 illustrates a step in the manufacture of micro-tubular elementsfrom micro-bore drawn filament;

FIG. 9 is a plan view of portion of an alignment plate for micro-tubularelements;

FIG. 9A, under the heading “pick and place”, is a plan (top) view ofalignment pocket, GNRs (micro-length tubes) in the pockets, wickingchannels, and tweezers;

FIG. 9B is a cross-sectional view of GNRs covered with stabilizingreagent and an absorbent pad approaching the alignment plate, while FIG.9C, similar to FIG. 9B, shows the absorbent pad positioned on thealignment plate and stabilizing reagent greatly reduced, and FIG. 9D,similar to FIG. 9C, shows the absorbent pad removed and tweezerspositioned at opposite ends of a GNR.

FIGS. 9E and 9F are plan (top) and vertical cross-sectional views,respectively, of a circular absorbent pad surrounding GNRs on a flatplate;

FIG. 9G shows an element being introduced with interference fit into achannel, the width of which is less than the diameter of the element,while FIG. 9H shows the element in a channel closed by a top layer;

FIG. 10 illustrates a centrifugal dryer cooperating with a drying andalignment plate to dry the micro-length tube elements;

FIG. 11 is, on enlarged scale, a diagrammatic representation of laserbeams ablating (removing or rendering in-active) selected regions ofcapture agent on end and inside surfaces of a micro-length tube element;

FIG. 12 is a view similar to FIG. 11, depicting the formation of a codeof capture agent on the inside surface of a micro-length tube flowelement;

FIG. 13 depicts a photo mask exposure scheme for forming a large laserbeam into beamlets that perform the steps of FIG. 11 or 12;

FIG. 14 is a diagrammatic top view of a system for placing discretemicro-length tube elements into open channels of a microfluidic assaydevice (see also FIG. 33);

FIG. 15 is a side view of a pick and place apparatus employing a tweezerinstrument for engaging end surfaces of the micro-length tube elements(see also FIG. 34);

FIG. 16 “PICK”, depicts the ends of tweezer tines approaching theoppositely directed end surfaces of a micro-length tube elementpositioned in the alignment plate;

FIG. 17 “PLACE with tweezer”, depicts the ends of tweezer tines leavingthe oppositely directed end surfaces of a micro-length tube elementpositioned in a flow channel of the microfluidic device;

FIGS. 18A, 18B, 18C, 18D and 18E represent, in large scale endcross-section views, a sequence of steps involved in placing and fixingthe position of micro-length tube elements in an assay device andcompleting the enclosure of a flow channel in the device;

FIGS. 19A and 19B illustrate the flow cross-sections of a flow channelwith micro-length tube element in place in a channel;

FIG. 20A is a diagram of a laser and computer controlled steering mirrorcutting a pattern in double sided PSA film with peelable liners;

FIG. 20B is a plan view of a laser-cut pattern in the material of FIG.20A, defining pneumatic control channels and features for integratedvalves and pumps while FIG. 20C is a magnified view of a portion of FIG.20B;

FIG. 20D is a vertical cross section, with parts broken away, on amagnified scale, of a microfluidic device comprising a reservoir layer,a pneumatic channel-PSA layer, a membrane, a fluidic layer containing aGNR in a fluidic channel and a glass layer, with a channel shunt formedin the membrane layer at the location of a stability bridge in thepneumatic channel-PSA layer, the membrane also closing a fluidic channeland containing a GNR placed in the channel (see also FIG. 56I);

FIG. 20E is a vertical cross section, with parts broken away, on amagnified scale, similar to FIG. 20D, except that the channel shunt isformed in the reservoir layer, the membrane also closing a fluidicchannel and containing a GNR placed in the channel (see also FIG. 56J);

FIG. 20F is a cross-section similar to FIGS. 20D and 20E, depicting avalve portion of the membrane deflected into a recess cut in thepneumatic channel-PSA layer, the membrane also closing off a fluidicchannel and containing a GNR placed in the channel;

FIG. 21 is a top-view of the fluidic sub-assembly on an enlarged scale(see also FIG. 103);

FIG. 22 is a perspective view of parts of the pneumatic sub-assembly asthe PDMS sheet comes together with the reservoir/pneumatic layer;

FIG. 23 is a plan view, looking up at the underside of thereservoir/pneumatic sub-assembly through its transparent PDMS membranesheet;

FIG. 24 is a plan view, again of the underside of thereservoir/pneumatic sub-assembly and the mating upper surface of theFluidic Layer sub-assembly;

FIG. 25 is a perspective view diagrammatically illustrating the matingaction of the two sub-assemblies with the micro-length tubes (e.g. GNRs)in the Fluidic Layer;

FIG. 25A is a side view illustrating the PDMS layer and the matingsurface respectively of the two subassemblies (Reservoir/Pneumatic Layerand Fluidic Layer) being pressed together with slight pressure;

FIG. 25B is a magnified view of a portion of FIG. 25A denoted by acircle in FIG. 25A labeled 25B;

FIG. 25C is a perspective view of the completed assembly viewed fromabove (as assembled with the glass layer facing up);

FIG. 25D is a perspective view of the completed assembly, viewed fromabove (after inversion, so that the reservoir layer faces up, the glasslayer faces down);

FIG. 26 is a top view of the completed assembly;

FIG. 27 is a schematic diagram in perspective of assembly steps for amicrofluidic assay device, a Figure very similar to FIG. 1 except fornumeral references instead of legends;

FIG. 27A is an exploded perspective view of the device of FIG. 27;

FIG. 28A is a perspective view on an enlarged scale of a fluidic channelof FIGS. 27 and 27A;

FIG. 28B is a further magnified view of a portion of FIG. 28A showingflow channels, hollow flow elements (e.g. GNRs), valve seats and pumpchambers;

FIG. 28C is an even more greatly magnified view of sets of extremelysmall hollow flow elements disposed in channels of FIGS. 28A and 28B(see FIGS. 5 and 7 for a representation of a single flow element);

FIG. 29 is a greatly magnified plan view of a portion of the channelstructure, showing two channels, with four hollow flow elements disposedin each (see FIG. 68) and indicating scanning;

FIG. 30 is a plan view of a single channel, with schematic illustrationof on-board pump and valve, and showing flow paths through and alongsidehollow flow elements;

FIG. 30A is Similar to FIG. 30, a plan view, but in greater detail, of amicrofluidic channel having a hollow flow element and a micro-pistonlocated between two micro-valves;

FIG. 30B is a cross section of the assembly of FIG. 30A taken on line30B of FIG. 30A;

FIGS. 30C, 30D and 30E are magnified views of portions of FIG. 30B asrespectively designated by circles in that figure, FIGS. 30C and 30Eshowing the membrane engaged upon a valve seat;

FIG. 30F is a view like the magnified cross-sections of FIGS. 30C and30E, except with the membrane deflected away from the valve seat;

FIG. 30G is like the magnified cross-section of FIG. 30D, except withthe membrane deflected;

FIG. 31 is a magnified diagrammatic cross section, with parts brokenaway of micro-fluidic channels of a device, and depicting the membranecapturing a hollow flow element in the channel, lines of flow beingindicated through and outside the flow element;

FIG. 31A is a view similar to FIG. 31 in which two layers (membrane andthe layer defining the side wall of the channels), both of PDMS, havebeen fused by covalent bonding to close the channels and secure thehollow flow elements;

FIG. 32 is a diagram of steps in the assembly process for the device ofpreceding figures;

FIGS. 32A, 32B, 32C and 32D are cross-sectional views of a microfluidicdevice through a hollow flow element, illustrating, diagrammatically,steps in employing PDMS surface activation and covalent bonding to formthe liquid-tight channels and secure the extremely small hollow flowelements in place in the channels;

FIG. 33 is a diagram in plan view of a pick-and-place instrumentpositioned above an X,Y translation table, a delivery plate fordiscrete, extremely small hollow flow elements and a receiving channelof multiplex micro-fluidic assay devices of the preceding figures (seealso FIG. 14);

FIG. 34 is a diagrammatic front view of a tweezer type pick and placedevice, and its support tower (see also FIG. 15);

FIG. 35 is a diagrammatic front view, similar to that of FIG. 34, of avacuum type pick and place device, and its support tower;

FIGS. 36 and 37 depict, respectively, picking, and placing side views ofa vacuum pick up device;

FIGS. 38, 39 and 39A depict a sequence of positions during placing of aflow element with a pick-and-place device, the + and − signsdiagrammatically illustrating the use of close-space electrostaticattraction between the channel wall and the element being delivered thatfacilitaties placement of the element and withdrawal of the tool;

FIGS. 39B and 40 illustrate element-securing and channel-sealing actionsoccurring during assembly of the device of preceding figures;

FIG. 40A, in plan view, illustrates hollow micro-particles (here,micro-length tubes) distributed in random fashion onto a flat surface,and a pick up head is shown;

FIG. 40B is a three dimensional diagram showing a picking head withvideo system and computer controller for effecting relative movements X,Y, Z and angular orientation theta between a surface element and thepicking head;

FIGS. 40C and 40D are side cross-sectional and plan views of a placementtool in form of a vacuum tip;

FIGS. 40E and 40F are side and horizontal cross section views of atweezer pick up tool;

FIG. 41 is a diagrammatic view, showing the repeated cycling duringmanufacture of a diaphragm valve formed by an overlying portion of aPDMS layer, which is bonded to the opposed structure at each side, thediaphragm valve, repeatedly closed with 3 psi positive pressure andopened with negative 8 psi pressure (vacuum), is found to overcome themolecular bonds being formed between diaphragm and valve seat, thus overtime, neutralizing the tendency for permanent co-valent bonds to formbetween contacting surface-activated surfaces, thus enabling thethus-formed valve to properly operate;

FIG. 42A is a diagrammatic showing of two opposed layers of PDMS,showing them in the natural state of the PDMS which is a hydrophobicstate with methyl group endings exposed;

FIG. 42B is a similar Figure following plasma oxygen plasma treatmentshowing the separated layers are terminated in OH groups;

FIG. 42C is a similar Figure illustrating permanent bonds between thehydroxyl groups producing oxygen covalent bridging;

FIG. 43A, similar to parts of FIG. 41, illustrates, in diagrammaticcross-section, a valve as initially assembled, comprising two opposedlayers of plasma-treated PDMS with a valve membrane portion of one layerdeflected, the opposed PDMS sheet forming an opposed valve seat;

FIG. 43B is similar to FIG. 43A following make and break process afterassembly;

FIG. 44A is similar to FIG. 43B, but shows each PDMS layer deflectedoutwardly from the other in the central region; and it illustrates tworegions of PDMS following plasma activation, steady contact in region R₂and cyclical contact and activation or separation in what is referred toas the valve region, R₁, illustrating the initiation of permanentbonding through the hydroxyls and the condensation reaction resulting inbridging oxygen in the region R₂, and in region R₁, where contact hadoccurred only temporarily and then removed, the surface having a numberof methyl groups or non-bonding or lower energy state species;

FIG. 45A, similar to FIG. 44A, is a figure illustrating a singledeflected surface opposing a planar surface.

FIGS. 44B, 43C and 45B show deflection chambers useful with,respectively, the devices of FIGS. 44A, 43A and 43B and 45A;

FIGS. 46A, 46B, 46C, 46D, 46E and 46F illustrate composite membranesuseful with the Make and Break process: FIG. 46A illustrates, incross-section, a composite membrane comprising a thin flexible sheet(PET) and a PDMS film of greater thickness; FIG. 46B illustrates, incross-section, a thin flexible sheet (PET) and a PDMS coating of lesserthickness; FIG. 46C illustrates, in plan view, a PET/PDMS laminationhaving circular stress relief channels formed in PET film; FIG. 46Dillustrates, in cross-section, a composite membrane similar to that ofFIG. 46A, but having stress relief slots formed in the thin flexiblesheet (PET); FIGS. 46E and 46F, similar to FIGS. 41 and 43C, illustratein cross-section a composite corresponding to that of FIG. 46D, inrespectively un-deflected and deflected states;

FIG. 47A is a view similar to that of FIG. 42B, but showing one layer ofPDMS with OH groups exposed and an opposed layer of silicon based rigidmaterials with OH groups exposed facing the PDMS layer;

FIG. 47B is a view similar to that of FIG. 42B, but showing one layer ofPDMS with OH groups exposed, and an opposed layer comprised of syntheticresin, carrying an intermediate bi-functional layer with OH groupsexposed facing the PDMS layer;

FIGS. 48A, 48B and 48 show a fluidic channel, FIG. 48, having twodifferent cross-sections, FIGS. 48A and 48B taken, respectively on thedetail lines A and B of FIG. 48, the details correspondinglyrespectively with previous FIGS. 44B and 45B;

FIG. 49B (taken on line 3 of FIG. 49A) and FIG. 49A show in respectivecross-section and plan views, a fluidic channel arrangement including achannel that extends from two inlets to an outlet, and, in communicationwith it, a zero dead volume sample channel; the cross section shows apneumatic chamber in which the membrane is shown in deflected openposition in solid lines, and closed by dashed line;

FIG. 50A shows, diagrammatically, a pneumatic tool (thereservoir/pneumatic layer) to which a fluidic layer is to be broughtinto contact and joined, the system capable of applying pressure orvacuum to the pneumatic tool, FIG. 50B being a magnified view of aportion of FIG. 50A;

FIG. 50C shows the pneumatic tool in cross-section with the fluidiclayer pressed against it, while FIG. 50D is a plan view, with theconnections to supply ports for selective application of vacuum andpressure to the pneumatic tool;

FIGS. 51A, 51B and 51C correspond respectively with the foregoing for asystem that is the same except the pressure controller is constructed toselectively apply vacuum at two values, −2 and −14 psi);

FIG. 52 is a view similar to FIG. 41, illustrating stages of the systemapplied to multiple valves simultaneously;

FIGS. 53A and 53B are graphs illustrating the selected pressures overtime and the development of properties of the contacting surfaces duringthe make and break process;

FIGS. 54A, 54B, 54C, 54D, 54E, 54F, 54G, 54H and 541 concern anothermake and break protocol having similarities with that of FIG. 41 to FIG.53B: FIG. 54A is a cross-section similar to FIG. 43C, but indicating anun-bonded area beyond the limit lines B of the pneumatic chamber, whileFIG. 54B is a magnified view of a portion of FIGS. 54A and 54C is a planview (top view), each denoting a leak path beyond the pneumatic chamber;FIG. 54D is a protocol flow diagram including cross-sectional viewsassociated with states within the pneumatic chamber during the make andbreak protocol; FIGS. 54E and 54F and 54G are views similar,respectively, to FIGS. 54A, 54B and 54C, but showing no leak path existsoutside the chamber boundary; and FIGS. 54H and 541 are similarrespectively to FIGS. 53A and 53B but have an initial phase usingconstant −3 psi deflection pressure, followed by positive and negativepressure cycling.

FIG. 55 pictures diagrammatically a pumping and valve state sequence bywhich liquid flow can be drawn into the piston from the left andexpelled to the right to produce a desired directional, pulsating flow.

FIGS. 56A, 56B, 56C, 56D, 56E, 56F, 56G, 56H, 56I, 56J and 56Killustrate functions performable by the membrane layer: FIG. 56A, closechannel; FIG. 56B, close channel, fix micro-particle in channel,particle shown as round in cross-section; FIG. 56C, close channel, fixmicro-particle in channel, particle shown of another shape; FIGS. 56D (alengthwise cross-section) and 56E (a transverse cross-section), closechannel, fix micro-particle element in the shape of a micro-length tubein channel; FIG. 56F, close channel, fix multiple micro-length tubes(e.g., GNRs) in channel, define flexible membrane for a valve and apiston; FIG. 56G, fix micro-length tube (GNR) in channel in fluidiclayer and define valves and piston that can be operated to constitute apneumatically-actuated membrane-pump; FIG. 56H, close channel and inconjunction with fixing micro-length tube in channel and definingflexible membrane of pneumatically actuated valve, form via for liquidto be pumped from or to reservoir; FIG. 56I, in conjunction with closingfluidic channel and fixing micro-length tube in the fluidic channel,bounding a pneumatic channel and forming a pneumatic shunt at blockageof the pneumatic channel; FIG. 56J, close channel, fix micro-length tubein channel, at other side, bound a pneumatic channel, pneumatic shuntformed in the reservoir layer about a blockage of the pneumatic channel;and FIG. 56K, in a fluidic layer of a device, close channel, fix tube inchannel and form membrane portion of micro-valve, and by exposed portionof membrane beyond the fluidic layer, define planar compliant surfacefor engagement with narrow lip of boss at pneumatic interface.

FIGS. 57 and 57A illustrate, respectively, two positions of amicrofluidic cartridge relative to a carrier plate upon which it isintended that the cassette be fixed while the plate is moved on aprecision X, Y stage relative to a fixed, finely focused opticaldetection system;

FIG. 57B is a cross-section view of the cartridge of FIGS. 57 and 57Anow fixed to the carrier plate on the movable stage, to move over afixed heater plate and optical detection system, the objective of whichis exposed to the cartridge through a hole in the heater plate. FIG. 57Bshows solenoid-actuated three-way valves 9 on moving X,Y stageselectively apply Pneumatic Conditions at Supply Ports defined by raisedbosses of FIG. 57C. Conditions supplied are: (1) atmospheric pressure,(2) positive (+) actuating pressure, (3) negative (−) actuatingpressure. Only connections to moving X, Y stage assembly are positiveand negative pressure line to manifolds feeding valves 9 and electricalcontrol lines for solenoid coils of the valves;

FIG. 57C is a magnified view of a portion of FIG. 57B;

FIG. 58 is an exploded view of the assembly of a bench top operating andscanning unit for scanning the microfluidic cartridge of FIGS. 57 and57A.

FIGS. 59A, 59B and 59C are plan views of the microfluidic and pneumaticchannel architecture of the cartridge of FIGS. 57 and 57A;

FIG. 60 outlines the fluidic architecture of a single microfluidicsubunit of the cartridge of FIGS. 57 and 57A, and, in tabular form,presents the steps of an immunoassay protocol conducted within thecassette (see also FIGS. 21, 28C and 103);

FIG. 61 diagrammatically illustrates the procedure of preciselydetermining the location of channels of a microfluidic cartridge, forinstance when fixed within the precise X, Y stage system of FIGS. 57,57A, 57B and 58;

FIG. 62 illustrates the fact that the precise position of channels, formonitoring, and the location of detection elements in the channel, forlater reading of results, can be accomplished in the same system;

FIGS. 63 and 64 are representations repeated in the later Scanningdrawings, illustrating signals obtained during position determination inthe absence of trace.

FIG. 65—General Schematic for Epi-fluorescent Scanning Microscope(similar to FIG. 101);

FIG. 66—Laser Beam Shape Isometric View;

FIG. 67—Laser Beam Shape Layout View;

FIG. 68—Micro-length tube element scan schematic;

FIG. 69—Acquisition Time Series;

FIG. 70—Overall Scan Sequence;

FIG. 71—Scan Sequence—Imaging;

FIG. 72—Scan Sequence—Discrete photo detector;

FIG. 73—Reading Code from Micro-length tube flow element;

FIG. 74—Preferred Implementation of previous Figures first block;

FIG. 75—Reading code and analyte quantity from micro-length tube flowelement simultaneously;

FIG. 76—Reading Code Consolidated;

FIG. 77—Bar Code in Micro-length tube Element;

FIG. 78—Scan Data File Snippet;

FIG. 79—Chip (microfluidic channel system) Layout;

FIG. 80—Find Channels ROI;

FIG. 81—Find Channels Scan Plot;

FIG. 82—Find Channels Data Segment Plot

FIG. 83—Find Channels Processing Flowchart;

FIG. 84—Find Elements ROI;

FIG. 85—Find Elements Scan Plot;

FIG. 86—Find Elements Processing Flowchart;

FIG. 87—Auto Focus Scan Plot;

FIG. 88—Auto-Focus Processing Flowchart;

FIG. 89—Auto-Expose Schematic;

FIG. 90—Auto-Exposure Procedure Flowchart;

FIG. 91—Laser/ROI Alignment;

FIG. 92—Fluorescence Scan ROI, bright field;

FIG. 93—Fluorescence Scan ROI, laser on, LED off;

FIG. 94—Fluorescence Scan Data, Full Scan;

FIG. 95—Fluorescence Scan, One Channel;

FIG. 96—Fluorescence Scan, One Element;

FIG. 97—Fluorescence scan data processing;

FIG. 98 depiction of a microfluidic system having microfluidic channelsand monitor locations;

FIG. 98A depiction of signals obtained in three phases at a set ofmonitoring positions under three different conditions, illustrating aproperly running assay;

FIGS. 98B and 98C, similar to FIG. 98A, depictions of signals obtainedduring improperly running assays;

FIG. 99 diagrammatically illustrates tracer signal during monitoring amicrofluidic channel at a single location, over a brief period of timeover which flow changes; time response at location 1: I. Pistonactuation (oscillating flow) @ full tracer concentration (no net flow);II. Pumping fluid with no tracer (in one direction, toward waste) todisplace tracer-laced fluid in channel; III. Oscillating flow, notracer:

FIG. 100 is similar to FIG. 99, but diagrammatically illustratesmonitoring a fluid to detect operation of a pump over many cycles ofproducing oscillating flow;

FIG. 101, similar to FIG. 65, illustrates, diagrammatically, therelation of a scanning system to a microfluidic device, shown alignedwith a channel at a region that does not contain a detection element;

FIG. 102 illustrates the cross section of the region of interest (ROI)of the optical system of FIG. 101 in relation to the microfluidicchannel and the cross-section profile of a fluorescence-exciting laserbeam;

FIG. 103 outlines the fluidic architecture of a single microfluidicsubunit of the cartridge of FIGS. 57 and 57A, and, in tabular form,presents the steps of an immunoassay conducted within the cassette;

FIG. 104 is a diagrammatic view of a microfluidic channel containingmicro-length tubes, on the insides of which are immobilized captureagents in the form of DNA (on some) and antibody (on others), whileFIGS. 104A and 104B are partially broken away cross sectional views of adevice implementing FIG. 104, taken at respective lines indicated inFIG. 104;

FIG. 105 is a similar diagrammatic view of a microfluidic channelcontaining micro-length tubes, on the insides of which are immobilizedcapture agents;

FIG. 106 is a diagrammatic view of four parallel microfluidic channelscontaining micro-length tubes, on the insides of which are immobilizedcapture agent; in channels 1 and 2 the capture agent is DNA and inchannels 3 and 4, the capture agent is antibody;

FIG. 107 is a is a diagrammatic view of four parallel microfluidicchannels containing micro-length tubes, on the insides of which areimmobilized capture agent, in channels 1 and 2 the capture agent is DNAand in channels 3 and 4, the capture agent is antibody, and with zonesheated at different respective temperatures;

FIG. 108 is a diagrammatic view of four parallel microfluidic channelscontaining micro-length tubes, on the insides of which are immobilizedcapture agent, each channel containing micro-length tubes, two havinginside surfaces functionalized with antibody and one functionalized withDNA;

FIGS. 109 and 109A are diagrammatic views from the end and side of amicro-length tube element being plucked by tweezers for removal from anopen fluidic channel of larger width than the element, using tweezersthe same as those shown respectively in FIGS. 18B and 21; and

FIG. 110 is a diagrammatic view from the end of a micro-length tubeelement being plucked from an open fluidic channel of smaller width thanthe element, using a tweezer the same as shown in FIG. 9G.

FIGS. 111 and 112 are line drawings showing the ambient analyte designspace, in accordance with embodiments of the present invention.

FIG. 113 is a graph of fractional occupancy vs. surface area, inaccordance with embodiments of the present invention.

FIG. 114 is a graph of a family of dose curves for fractional bindingand sample volume dependence, in accordance with embodiments of thepresent invention.

FIG. 115, illustrations (a)-(d), are graphs of dose curves for fourdifferent antigens, in accordance with embodiments of the presentinvention.

FIG. 116 is a graph of RFU signal vs. reaction volume, in accordancewith embodiments of the present invention.

FIG. 117 is a top view of eight fluidic circuits, in accordance withembodiments of the present invention.

FIG. 118 is a top view of an exploded view of one fluidic circuit, inaccordance with embodiments of the present invention.

FIG. 119 is a graph of fluorescent signal vs. time for a sample mixingexperiment, in accordance with embodiments of the present invention.

FIG. 120 is a graph of fluorescent signal vs. volume of buffer inreservoir for a sample mixing experiment, in accordance with embodimentsof the present invention.

FIG. 121, illustrations (a)-(d), are side views four GNRs at differentlengths and the associated GNR internal coating density curves, inaccordance with embodiments of the present invention.

FIG. 122 is a flowchart of an assay cartridge creation process, inaccordance with embodiments of the present invention.

FIG. 123 is a flowchart of a process for running an assay, in accordancewith embodiments of the present invention.

FIG. 124 is a top view of a portion of the assay cartridge, showingfluidic and pneumatic channels, in accordance with embodiments of thepresent invention.

FIG. 125 is a top view of a portion of the assay cartridge showing thepneumatic channels, in accordance with embodiments of the presentinvention.

This application is a stylistically edited version of a correspondingPCT application which has been incorporated by reference. The edits haveincluded some changes to the figure designations. A table of the newdesignations and corresponding figure designations in the earlier PCTapplication publication is given below. Any use of the old designationin the text of this application, if it appears, should be referred tothis table for identifying the figure (or numeral) intended.

PCT Publication Current Application FIG. 50H (Sheet 12) FIG. 9H FIG.50G(i) (Sheet 12) FIG. 9G FIG. 33A′ Numeral 12B (Sheet 42) FIG. 30BNumeral 30C FIG. 33A′ Numeral 12C (Sheet 42) FIG. 30B Numeral 30D FIG.33A′ Numeral 12D (Sheet 42) FIG. 30B Numeral 30E FIG. 51 A-11 (Sheet 66)FIGS. 46C, 46D, 46E, and 46F FIG. 51A-12 (Sheet 67) FIGS. 47A and 47BFIG. 51B(ii) (sheet 68) FIGS. 51B, 51C and 51D FIG. A-16 Detail A (Sheet69) FIG. 48A FIG. A-16 Detail B (Sheet 69) FIG. 48B FIG. 51A-18 (Sheet70) FIG. 49A FIG. 51A-19 (Sheet 70) FIG. 49B FIG. 52A1 (Sheet 71) FIG.54A FIG. 52A1_(m) (Sheet 71) FIG. 54B FIG. 52A plan (Sheet 71) FIG. 54CFIG. 52 A 1N (Sheet 72) FIG. 54D FIG. 52A2 (Sheet 73) FIG. 54E FIG.52A2_(m) (Sheet 73) FIG. 54F FIG. 52A2 plan (Sheet 73) FIG. 54G FIG.52A2N (Sheet 74) FIGS. 54H and 54I FIG. 53 Drawing A (Sheet 76) FIG. 56AFIG. 53 Drawing B (Sheet 76) FIG. 56B FIG. 53 Drawing C (Sheet 76) FIG.56C FIG. 53 Drawing D (Sheet 76) FIG. 56D FIG. 53 Drawing E (Sheet 76)FIG. 56E FIG. 53 Drawing F (Sheet 76) FIG. 56F

DETAILED DESCRIPTION Description

One of the problems addressed concerns the surface area associated witha micro-length tube element, i.e., an element having length less than700 micron and a micro-bore diameter between about 75+/−50 micron thatis fixed in a flow channel and exposed to flow of liquid sample, e.g., aglass nano reactor “GNR” Such devices are typically made of endlesslydrawn micro-bore filament such as used to form capillary tubes, but inthis case, the filament is finely chopped in length to form discrete,shorter micro-flow elements. It is realized that capture agentimmobilized on the surface of such a device, applied by immersiontechniques, can raise a significant depletion problem. This occurs, forinstance, when attempting to characterize concentrations of an analyteat low levels such as a few pico-grams per milliliter, as is desired.The phenomenon referred to as “depletion” occurs in which theconcentration of analyte in the sample being measured can bedisadvantageously depleted volumetrically as a result of binding to alarge active area of the flow element. This results in reduction ofsensitivity of the assay, and therefore its usefulness. To explainfurther, any analyte in an ELISA or sandwich type of amino assay onantigen will bind to a capture antibody in a way that is governed by akinetic reaction, a dynamic process. While analyte such as an antigenbinds to capture agent such as an antibody, the reverse also occurs, thebound analyte molecules unbind from the capture agent. The kineticsconcern an “on” rate and an “off” rate—analyte being captured andanalyte being released. The capture reaction will continue, depletingthe analyte in the ambient volume, and reducing its net rate of capture,until the system reaches equilibrium in which the rate of binding isequal to the rate of unbinding. The gradual action occurs according to asubstantially exponential curve.

The absolute value of the equilibrium condition depends on the originalconcentration of the analyte in the volume of sample being assayed.Increase in concentration results in a higher signal, decrease inconcentration results in a lower signal. In cases in which assaydepletion occurs, the concentration of the analyte in the sample isdetrimentally decreased over time. It is realized that micro-lengthtubes fixed in flow channel may present an excess of capture agent inthe volume of liquid sample to which the element is exposed, decreasingthe effective concentration of the analyte. The concentration decreasesat an excessive rate, relative to initial, starting point concentrationsought to be measured. While efforts to calibrate for this are helpful,such depletion ultimately lowers the sensitivity of the assay because,as the signal goes down r, it approaches the noise level, and results ina lower signal-to-noise ratio, i.e. an inherent reduction ofeffectiveness of the assay. (Already there are significant contributorsto noise i.e., background, nonspecific binding of capture antibody,fluorescence noise, electronic noise, etc.). Therefore, especially fordetecting small concentrations, it is desired not to deplete the initialvolume of the analyte in manner that does not contribute positively tothe assay measurement. Efficient ways to do that, as by somehow limitingthe amount of exposed surface have not been apparent. This may be seenas an inherent problem with use of micro-flow elements of variousdescriptions that are coated by immersion or the like and used in animmunoassay or sandwich assay or even a molecular diagnostic type ofassay. One typically wishes to immerse the elements in capture agent,e.g. an antibody or some type of moiety that is a capture molecule forthe analyte to be sensed or detected, to uniformly coat all surfaces ofthe element. One object of invention is to overcome this problem withrespect to micro-length tube elements characterized by an inside surfaceand an outside surface, or often also with two end surfaces. Adding upall surface area over which a density of capture molecules is coated canadd up to a surface area on the order of over 100,000 square microns.This is the case for a preferred form of micro-length tube, having onthe order of about: a length of 200 microns, an external diameter orwidth of 125 microns, and an internal diameter or width of 70 microns. Aparticular problem addressed here is to find practical approaches foraccurately reducing active surface area of immersion-coated flow assayelements in general, and in particular, micro-flow elements, and inparticular micro-length tube elements.

A further problem being addressed here concerns treatedmicro-flow-elements that are to be in fixed positions in channels forexposure to flow of sample. It is desirable to expose the elements inbatch, in free state to an immobilization process for applying thecapture agent or antibody to the element surface, and then transfer eachelement mechanically to its fixed position in a channel, for instance ina channel of a multiplex micro-fluidic “chip” (or “cassette”). It isdesired to use a quick and accurate placement process, for instance apick and place device mounted on an accurate X, Y stage. For suchpurpose, it is desirable to physically contact the tiny element forpicking it up from a surface and placing it in an open channel, which isthen closed to form a micro-fluidic passage. It is desirable to employgrippers, e.g. a tweezer instrument that contacts the outer surface ofthe device. The pick and place action is made possible by pre-aligningopen channels to receive the micro-flow elements and the surface onwhich the free elements are supplied with the automated pick-and-placeinstrument. This enables the grippers to pick up and place themicro-flow elements precisely in desired flow channel positions in whichthey are to be fixed. We recognize a problem arises with having anactive capture agent, e.g. antibody, immobilized on outer surfaces of anelement. Such a coating is susceptible to mechanical damage as a resultof the mechanical manipulation process. Outside surfaces of micro-flowelements come in contact with (a) a supply surface, e.g. an aligningpocket or groove, (b) the transferring grippers, and (c) surfaces of thechannel in which it is being deposited. All of these contactsopportunities give rise to possible damage to the fragile coated captureagent, which typically is a very thin layer of antibody or the likeadsorbed to the surface of the flow element. This coating is often onlya few molecules thick, thickness of the order of nanometers or tens ofnanometers, and is quite fragile. The net result of damaging a capturesurface of the placed micro-element is seen during read out of theassay. If the surface has been scratched or perturbed in any way, thatcan give rise to an irregular concentration or presentation of capturedanalyte, the signal can be irregular, and contribute toirreproducibility or poor performance of the assay.

We thus realize it is desirable not to have immobilized active captureagent on the outside surface of a micro-flow element, and especiallymicro-length tube element, where it is susceptible to damage and whereit contributes to increasing the total surface area of the capture agentor antibody that contributes to depletion.

The features described in the claims and hereafter address these andother important problems.

Discrete micro-flow elements are immersed in liquid containing captureagent, such as antibodies or antigens, and, after coating by the liquid,are picked, and placed into channels for flow-through assays. Themicro-flow elements are in preferred form of discrete micro-lengthtubes, defined as micro-flow elements of length less than about 700micron, and bore diameter of 70+/−50 micron. The flow elements aresurface-treated so active capture agent, e.g. capture antibody, is noton the outside, or is of limited outside area. For this effect,micro-flow elements, or in particular, micro-length tubes, are disposedin a bath of active agent and violently agitated, resulting in coatingof protected inside surface, but due to extreme shear forces, a cleanarea on the outside surface, for instance the entire outside cylindricalsurface of a round cross-section discrete micro-length tube. In lieu ofor in addition to this shear procedure, a special filament-manufacturingprocess is conceived that results in preventing coating an exteriorsurface of flow elements with a predetermined capture agent. Captureagent on selected coated areas are ablated or deactivated with preciselypositioned laser beam, such as can be produced by a mask forsimultaneous treatment of a large number of elements, leaving residualactive agent of defined area on the inside surface of micro-flowelements. Residual capture agent, itself, on the inside of the elements,usefully defines a readable code related to the desired assay. Flowchannel shape is sized relative to flow elements fixed in the channel toallow (a) bypass channel flow along the exposed outside of a micro-flowelement to reach and flow through later elements in the channel in caseof clogging of the first element, along with (b) sample and assay liquidflow through the micro-flow element to expose the surface to captureagent and other assay liquids. Lacking the need to attempt to seal theoutside, the element can simply be gripped, as by an elastomeric sheetpressed against the element. Electrostatic attraction between flowelement and channel wall is employed to fix the element in position,overcoming any disturbing force of the placing instrument as it is drawnaway after delivery of the element. After assay, fluorescence is excitedand read by special scanning confined to micro-flow element geometry.Locators are seeded in the recorded data, and used to locate the regionsof interest in detected fluorescence data, e.g. from micro-length tubes.Code, written with the capture agent substance inside the micro-flowelement is read through a transparent wall of the element. Efficientassembly and tooling features are disclosed. All features are applicableto micro-length tubes, enabling their efficient use. A number of thefeatures are or will be found to be useful with other hollow elements,for example, longer micro-flow elements.

In respect of scanning, the purpose of this invention to deliver amethod for performing a fluorescence measurement of multiple immobilizedelements contained in a microfluidic chip. This method provides fordetermining the paths to be followed during the scanning, as well as theproper focus, and camera exposure. The method is based on a knowngeneral chip layout. The method provided results in the ability to placethe chip to be measured into the scanner and then start the scan withoutany additional manual settings required. The method does the rest, andproduces the desired fluorescence measurements as the results.

Certain aspects of invention involve eliminating or preventing theoccurrence of active capture agent on outside surfaces of micro-flowelements, e.g. extended outside cylindrical surface, and/or endsurfaces, while leaving active capture agent on the inside surfaceunperturbed, or of a desirable area or pattern. Features addressing thisaspect include techniques to selectively limit the capture agent on theinterior surface and steps that act in combination with outside andinside surfaces to achieve the desired result.

For the specific advantage of reducing the overall capture surface area,two aspects of invention will first be described, and the effect oftheir combination. A first technique is employed to eliminate or preventcapture agent, e.g. antibody, from immobilizing to the outside surfaceof hollow flow elements, especially, micro-length tube elements. That isdone during a batch coating process, and involves suspending discretehollow elements, especially micro-length tube elements, in an Eppendorfftube or other tube with the capture agent of interest and aggressivelyagitating fluid to impart disrupting shear forces to the exteriorsurface of the elements. Preferably, this is achieved by vortexing thefluid at high speed, for instance employing an instrument that orbitsthe container at approximately 2000 rpm of the orbiter, about an orbitalpath of the supporting shaft of diameter of about 25 mm.

The micro-tub elements are placed with a volume, e.g. a milliliter ofcapture agent, e.g. antibody. The appropriate vortexing speed, isdependent e.g. on the nature of the suspension, e.g. the viscosity ofthe liquid chosen, and can be easily determined experimentally. It isset by observing whether the capture agent is effectively non-existenton the outside, long surface of the micro-length tube elements, e.g. theoutside cylindrical surface in the case of the body being of circularcross-section.

The physical principle involved concerns shearing force on the outsidesurface of the micro-length tube element that acts to prevent binding ofthe capture agent to the surface through an adsorption process. One canobserve whether the vigorous agitation is sufficient to shear off anycapture agent, e.g. antibody that has already been bound to thatsurface. At the same time, the inside surface is environmentallyshielded from this shearing by virtue of the geometry which is tubular,and the micro-size of the bore of the tube. This prevents vortexing fromcausing any turbulence to occur within the element. Only laminate flowconditions exist. With micro bore elements the Reynolds number is alwayslow enough to ensure that that laminar flow condition exists on theinside surface. Under these conditions, the velocity of fluid traversingin the micro-length tube element at the interior wall interface is bydefinition zero. So there is no shear force involved there, whereas theoutside is in a highly turbulent, high shear force environment.

The observed result of aggressive agitation, e.g. vortexing, is thatfluorescence which is observed by performing a sandwich assay iscompletely absent from the outer cylindrical surface of a micro-lengthtube element, whereas it is present in an observable way on the insidesurface. In the case of square-end micro-length tube elements,fluorescence is also present on the end faces of elements.

Vortexing is the presently preferred technique for producing the shearforces. The case showed here employs orbitally rotating the micro tubein a very rapid manner back and forth in small circles at a rate ofapproximately a couple thousand rotations per minute, and an excursionof about 25 mm.

However, any type of rapid oscillation that creates a high degree ofturbulence can be employed, so a back and forth motion, a circularrotation, anything that would very rapidly mix the fluids and createhigh shear forces will suffice.

In summary, micro-length tube elements in the presence of aggressiveagitation leads to removal of capture agent, e.g. antibodies, fromoutside surface of the elements, and prevention of their coating withthe agent, but leaves the inside surface of the micro-length tubeelement in condition to immobilize capture agent, e.g. captureantibodies, for subsequent interaction with analyte of the sample.

As an alternative to the high shear technique, we conceive an alternateprocess in which, during the original drawing of the small bore tube,and prior to the point along the draw path that the usual removableprotective polymer coating is applied to the filament, that a non-stickcoating, e.g. sputtered gold, silver or graphite, is applied to thefilament, e.g. by passing through a sputtering chamber. Silane orsimilar coating must be applied to receiving surfaces before captureagent, e.g. antibodies will attach. However, due to the properties ofthe sputter coating, or the like, the surface will not receive thesilane or equivalent, then likewise, the active capture agent.

Another feature of invention concerns realizing the desirability andtechnique of removing coated capture agent from selected end surfaces ofthe flow elements and a margin portion or other portion of the interiorsurface. Preferably, following the aggressive agitation process, themicro-length tube elements are further processed using a laserelimination process that removes or de-activates capture agent, e.g.antibodies, from surface from which the agent was not removed by thehigh shear process. Those surfaces include transverse end surfaces and aselected portion of the inside surface, leaving only an annular stripeon the inside surface sized sufficient to process the assay, but smallenough to reduce depletion of the analyte from the sample.

In a preferred form an ablating laser is arranged transversely to theaxis of elongation of the micro-length tube elements with the effectthat the energy arrives though parallel to the end faces has aneutralizing or removal effect on the capture agent that is on those endfaces, as a result of incidence of substantially parallel radiation, butalso of internal reflection scattering of the radiation by thetransparent substance that defines those end faces.

The net effect of two novel processes described, if used in novelcombination, is to leave only a band of selected dimension, which can besmall, of capture agent immobilized on the inside surface of themicro-length tube element. This can be done in a way that leaves one ormore bands separated by a space of no capture agent. Thus one cangenerate a single band in the center or a single band closer to one endor multiple bands distributed along the length of the micro-length tubeelement. These bands can be of different widths, can have differentspacing, and can be of the form of a code, e.g. a bar code, which isuseful to encode the particular flow element, e.g. micro-length tubeelement.

Further is a description of manufacturing techniques that have importantnovel features.

The micro-length tube elements are first cut, i.e. chopped, frompreviously supplied continuous small-bore filament into the short,discrete micro-length tube elements. They are then treated in batchmanner.

A bulk of the micro-length tube elements is then exposed in anEppendorff tube to wash buffer. After washing processing is performed,the buffer is removed, and replaced with a silane. By use of thissimple, low-cost immersion step, the silane is allowed to bind to all ofthe surfaces of the micro-length tube elements. Excess silane after aperiod of time is washed away with water in a buffer. Then a captureagent, e.g. antibody, in solution is added to the Eppendorff tube withthe bulk of micro-length tube elements and allowed to incubateovernight. The incubation is performed on the orbital vortexer forapproximately 16 hours at 2000 rotations per minute, with of the orderof one-centimeter diameter orbital motion. The orbital plate thatcontains the numerous Eppendorff tubes is approximately 6 inches indiameter, but the orbital motion is a circular pattern counterclockwiseand then clockwise motion in a circular pattern of diameter ofapproximately 2 centimeters.

After the vortexing process is completed, the net result is that thecapture agent has been immobilized on the inside surface of themicro-length tube element and also on the end faces but it is notpresent on the outside cylindrical surface of the tubular element. Thecapture gent solution is removed from the Eppendorff tube, which isreplaced with a wash buffer, a wash buffer solution, and the wash buffersolution is then further replaced with a stabilizing buffer, what wecall a blocking buffer. In the preferred embodiment, a commercialmaterial called StabilCoat® solution is used.

StabilCoat® blocking solution is introduced to the Eppendorff tube alongwith micro-length tube elements, then a portion of those elements isaspirated in a pipette along with some of the StabilCoat®, and dispensedonto an alignment plate. The alignment plate contains a series ofrectangular shaped pockets, each designed to accommodate and position asingle micro-tube element within a small space, preferably withclearance tolerance sized in microns, a space of 10 to 50 micronsbetween the micro-length tube element and the walls of the pocket. Afterthe elements are allowed to roam on the plate, they fall into thesepockets still in the presence of the solution of the buffer solution.The excess buffer solution is removed from the alignment platecontaining the micro-length tube elements by placing their plates withelements into a centrifuge or centrifuge holder and centrifuging atapproximately two thousand rpm, for 30 seconds, thereby removing allexcess StabilCoat® solution from the plate and the micro-length tubeelements. This process is facilitated by the novel design of the plateshown, in which drain channels extend radially from the pockets.

The process of creating immobilized antibodies or other activebiological species to serve as assay capture agent on micro-particles,and particularly the inside of hollow glass micro-particle elements, andthen transferring the functionalized micro-particles to the channel of amicrofluidic device, according to the invention, involves a number ofcritical steps. Long spools of continuous capillary tubing ofapproximately 125 micron OD, 70 micron ID, with a few microns thickpolyamide protective coating on the exterior are obtained from amanufacturer. Typically these are drawn, fiber-like filaments, withhighly polished and accurately dimensioned inner and outer surfaces. Thespools are rewound onto a mandril in a tight mono-layer wrapping fashionsuch that each turn or strand, wound around the mandril is in contactwith adjacent strands. The wrapped series of strands consists of forinstance a hundred strands. It is then wrapped with an adhesive tape bywhich the strands are captured in an assembled unit. The tape is thenslit parallel to the mandrill axis and the tape removed bringing thestrands with them. This produces a thin linear array of monofilaments inclose contact with one another. The relatively long array ofmonofilaments is then presented to a wafer dicing saw (as used in thesemiconductor industry for dicing thin ceramic wafers), the filamentsare diced at repeat distances of approximately 250 microns, producingcylindrical tubular micro-particles of that length.

After the dicing process, the individual micro-particle elements stillretained on the tape are liberated using a hot aqueous detergentsolution which liberates the individual elements from the tape. They areallowed to settle into the bottom of a beaker, the tape is removed fromthe solution, then the aqueous solution is removed and replaced with ahot sulfuric acid and peroxide solution, used to dissolve the polyamidecoating from the outside surface of the glass elements. This is followedby a substantial washing cycle, a number of flushings with de-ionizedwater to remove the residual sulfuric acid solution. After the hollowglass elements have been thoroughly washed, they are silanized using asilane reagent such as APTES which stands for aminopropyltriethoxysilane(“silane”). The micro-particles are allowed to incubate in the silanesolution for approximately an hour after which they are rinsed and curedin an oven for another hour after which they're then stored in anethanol solution. To ensure the silane reaches the inside surface of thetubular micro-particles, vigorous vortexing is used to uniformlydistribute the silane throughout the interior of the hollow elements.The micro-particles are then transferred to a reagent solutioncontaining the capture molecule of interest, for example a captureantibody.

For this purpose the silanized micro-particles are transferred to a vialcontaining the capture agent for example a capture antibody. The captureantibody is allowed to bind to the active silane surface for a period of16 to 24 hours after which a rinsing cycle is performed to remove anyloosely bound capture agent and then finally the functionalizedmicro-particle elements are transferred to another vial containing astabilizing compound such as SurModics' brand StabilCoat®. They remainin the StabilCoat® solution until it is desired for them to betransferred to a microfluidic device, such as a microfluidic cartridge.They are stored in the StabilCoat solution in a refrigerator until readyto be used.

Thus, after the immobilization process is complete wherein the surfaceof the micro-particle, or inside surface of the hollow glassmicro-particle elements have the active species immobilized to thesurface, the particles are re-suspended in a stabilizing compound suchas StabilCoat (trademark of SurModics, Inc.) for storage until needed.The purpose of the stabilizing compound is to protect the activity ofthe immobilized species when the micro-particles are taken out of thereagent and exposed to atmosphere. We have discovered that theantibodies immobilized to a surface without a protective coating, duringstorage, have a tendency to degrade in their functionality (becomepartially denatured) with the result of a higher coefficient ofvariation of assay execution, severely affecting the precision(repeatability) and sensitivity of the assay, and thus preventing anaccurate quantification assay.

The stabilizing reagent consists of high concentrations of sugars andproprietary compounds. We have found that when the water component isallowed to evaporate, thick residue of this sugary compound is leftbehind, which under low humidity conditions has a tendency tocrystallize and become a fairly rigid structure which can causeparticles in this compound to become almost irreversibly stuck to anysurface that it comes in contact with and has been allowed to dry in.

According to a preferred step in the process of moving themicro-particles from the liquid state to the dry state involvesdispensing the micro-particles in a solution of the protective coatingliquid, e.g., StabilCoat, onto an alignment plate such as a preciselymicro-machined grooved pocketed plate such as a silicon micro-machinedplate with pockets configured to accept the micro-particles in an arraypattern. The shaped pockets are, e.g., rectangular in the case of shortsegments of capillary tubing forming hollow micro-particle elements. Theexcess liquid compound is either spun off in a centrifuge or wicked awayusing an absorbent pad, leaving behind a small residue of protectivecompound about the micro-particle, e.g., both inside the hollow glassmicro-particle element and around the outside of the element in thepocket capturing the element.

We have discovered that allowing the stabilizing compound to dry in anenvironment of relative humidity less than approximately 60% relativehumidity has a deleterious effect of retaining the micro-particlesthrough a sugar crystalline structure that bonds the elements into thepocket. It has been found maintaining the humidity of 55 to 60% relativehumidity or higher softens (hydrates) the stabilizing compound to thepoint at which the viscosity approaches nears that of water enabling apick and place process to proceed for placing the micro-particles inchannels of a microfluidic device. Under these conditions, one may useautomated tweezers or a vacuum picking head to grab the micro-particlesout of the capturing pockets and place them into their finaldestination, a microfluidic channel of a microfluidic device.

The steps of assembly of the micro-particles is summarized as follows.

When ready to assemble the pick and place technique previously describedin U.S. Application No. 61/608,570 and Ser. No. 13/427,857 is employedin which the micro-particles are placed in a groove locator plate.

There is, however, an alternative way that this could be done. Thisinvolves distributing the micro-particles in random fashion onto a flatsurface not having grooves or alignment pockets. The advantage of thisprocess is not requiring a micro machined component for themanufacturing process. The disadvantage is that the micro-particles arerandomly distributed in a pile. The tendency is for them to come to restin a monolayer on the surface, but with random orientation. Further, asa result of removing the excess StabilCoat by centrifuge or wicking awaythe excess stabilizing solution, it has been observed that themicro-particles have a tendency to agglomerate into dense pack of therandomly oriented elements.

We realize a solution exists to this problem. Individual micro-particlescan be picked from this dense pack by use of a placement tool, e.g., avacuum tip, that engages the top surface of the elements, in combinationwith a vision system used to identify an individual element and a motionsystem responsive to the vision system, which orients the relativerelation of the vacuum pickup tip and the micro-particles in both X andY coordinates, and angular orientation. The placement tool can be movedin minute movement, slightly laterally within channel, to bring GNRagainst one side wall. Preferably a table carrying the fluidic layer,preferably channel side up, moves in computer-controlled X, Y and Z, andthe placement tool is stationary, with only the grippers (e.g. tweezers)moving under computer control.

CyVek temporarily secures the micro-particles in open channels prior tothis fluidic component with open-sided channels being turned upside downfor bonding to the flexible membrane. The membrane is carried by thepneumatic component of the cartridge, to complete the assembly.

Currently this is done in over-width, under-depth open micro channels.Electrostatic attraction draws the GNRs from the placement tool(enabling tool withdrawal) and holds them against one side of theover-sized channel with sufficient certainty that the assembly can beoverturned for bonding against the membrane. Compressive force of theelastically compressible membrane permanently then fixes the GNRspermanently in position.)

An alternative technique is contemplated that would avoid need for theplacement tool to move laterally against one side of the over-sizechannel. In this case the open channel in a resilient PDMs (siliconerubber) channel-defining layer is slightly undersize in width and may beoversize in depth relative to the GNR.

In this case the placement tool thrusts the GNR down with force-fit intothe channel, and the sides of the channel are slightly, resilientlydeformed to accept the GNRs. The sides then grip the micro-particlestightly. The GNRs may even be thrust so deep into the channels that theyare submerged below the face plane of the fluidic layer.

Final assembly then proceeds: the fluidic layer is turned upside downand bonded to this up-facing membrane.

In a typical system the GNRs are short segments of fine capillarytubing, e.g., outside diameter, e.g., 125 micron, inside diameter 70micron and length 250 micron).

The accepting microfluidic channels in one instance are (uniquely) widerthan the micro-particles, and shallower. Successful placing depends uponelectrostatic force associated with silicone rubber (PDMS, an electricinsulator material) to attract the micro-particles from the placinginstrument, and retain them in position during the completion of theassembly process, which even involves turning the over-size channelsupside down. In this case elastic deformation of the covering membraneat the sites of the raised top surface of the micro-particlespermanently fixes the location of the micro-particles.

In alternative technique, the microfluidic channels are undersize,widthwise, relative to the micro-particle width, and the placing toolforces the micro-particles into the channels, to obtain a mechanicalgrip by elastomeric material forming the sides of the channel. Again,the microfluidic channels can have depth less than the micro-particles,so that the membrane stretches over them to further fix their location.In another instance, the channel depth may exceed the depth of themicro-particles, and the placement submerges the particles such that theoverlying membrane is not locally disturbed by the presence of themicro-particles.

The novel process of creating immobilized antibodies or other activeassay capture agents on micro-particles, and particularly on the insideof hollow micro-particle elements (micro-length tube elements) will nowbe described by reference to a specific example. This will be followedby examples of novel transfer of functionalized micro-particles to anoperative position within a microfluidic device, for instance to amicrofluidic channel.

In the preferred case of micro-length glass tube elements, long spoolsof continuous glass capillary tubing of approximately 125 micron OD, 70micron ID, with a few microns thick polyamide protective coating on theexterior are obtained from a manufacturer. Typically these are drawn,fiber-like filaments, with highly polished and accurately dimensionedinner and outer surfaces. The spools are rewound onto a mandrel in atight mono-layer wrapping fashion such that turns or strands woundaround the mandrel are in contact with adjacent strands. The wrappedseries of strands comprises, for instance, one hundred strands. Thestrands are then wrapped on their outside with an adhesive tape by whichthe strands are captured in an assembled unit. The tape is then slit atone point parallel to the mandrel axis and the tape with the strands isremoved. This produces a thin linear array of monofilaments in closecontact with one another. The relatively long array of monofilaments isthen presented to a wafer dicing saw (as used in the semiconductorindustry for dicing thin ceramic wafers). The filaments are diced atrepeat distances of the order of 1000 micron or less, to producemicro-length tube elements. Preferably, the repeat distances are lessthan 700 micron, and in preferred instances, of the order of 250microns, producing cylindrical micro-length glass tube particles of thatlength. Those tubes having internal volume of the order of a nanoliterand are referred to as “glass nano reactors”, or “GNR”s).

After the dicing process, the individual micro-length particles orelements, still retained on the tape, are liberated from the tape usinga hot aqueous detergent solution. They are allowed to settle into thebottom of a beaker and the tape is removed from the solution. Then theaqueous solution is removed and replaced with a hot sulfuric acid andperoxide solution, to dissolve the polyamide coating from the outsidesurface of the glass elements. This is followed by a substantial washingcycle, employing a number of flushings with de-ionized water to removethe residual sulfuric acid solution. After the hollow glass elementshave been thoroughly washed, they are silanized using a silane reagentsuch as APTES (aminopropyltriethoxysilane, “silane”). Themicro-particles are allowed to incubate in the silane solution forapproximately an hour. To ensure the silane reaches the inside surfaceof the tubular micro-particles, vigorous vortexing is used to uniformlydistribute the silane throughout their interior to form a silanecoating. The micro-length tubes are then rinsed and cured in an oven foranother hour after which they are stored in an ethanol solution, readyfor being functionalized, (i.e., treated to surface-immobilize a capturemolecule of interest, for example a capture antibody).

For functionalizing, the silanized micro-particles are transferred to avial containing the capture agent, for example a capture antibody.Again, to ensure the capture agent reaches the inside surface of thetubular micro-particles, vigorous vortexing is used, which is found tobe capable of substantially uniformly distributing the capture agentthroughout the tubular interior of the micro-length particles to producesubstantially uniform immobilization of the capture agent over thelength of the interior surface. The capture antibody under theseconditions is allowed to bind to the active silane surface for a periodof 16 to 24 hours. Advantageously, as herein further described, it isfound, that the vortexing conditions of the process, preventimmobilization of the capture agent to occur to longitudinally-extendingouter surfaces of the violently agitated micro-length particles.

After the immobilization process is complete, with inside surfaces ofthe hollow elements carrying a substantially uniform coating ofimmobilized active species, the functionalized micro-particle elementsare re-suspended by transfer to another vial. The vial contains astabilizing compound such as SurModics brand StabilCoat′. We havediscovered that antibodies or other biological capture agentsimmobilized to a surface without a protective coating, during storagehave a tendency to degrade in their functionality (become partiallydenatured) with the result of a higher coefficient of variation of assayexecution. This can severely affect the precision (repeatability) andsensitivity of many assays, and thus prevent quantification to thedesired degree. Thus the purpose of the stabilizing compound is toprotect the activity of the immobilized species when the micro-particlesare taken out of the reagent and exposed to ambient conditions.

The functionalized micro-length tube elements remain in the stabilizingsolution stored in a refrigerator until ready to be used, i.e. until itis desired for them to be transferred to a dry state within amicrofluidic device, such as a microfluidic cartridge.

A preferred step in the process of moving the micro-particles from theliquid state to the dry state involves dispensing the micro-lengthparticles in a solution of the protective coating liquid, e.g.,StabilCoat, onto a so called “pick-up plate”, for presenting themicro-particles for subsequent picking and placing in microfluidicchannels. A preferred example of a pick-up plate is a preciselymicro-machined grooved locator plate, preferably a pocketed locatorplate such as a silicon micro-machined plate, with pockets configured tocapture individual micro-particles in an array pattern. The shapedpockets are, for example, rectangular in the case of short segments ofcapillary tubing forming hollow micro-particle elements (micro-lengthtubes). The excess liquid compound is either spun off in a centrifuge orwicked away using an absorbent pad, as described herein. Eithertechnique leaves behind a small residue of protective compound about themicro-particle, e.g., both inside the hollow glass micro-particleelement and around the outside of the element, i.e. in the pocketcapturing the element. In cases later described in using a planar pickup plate, a small residue remains with the elements in a puddle on theplane surface on which the elements are displayed. In mass manufacture,it is typically desired, after applying the micro-particles to a pick upplate, to store the plate and particles prior to installation into themicrofluidic device.

We have discovered that allowing the stabilizing compound to dry in anenvironment of relative humidity less than approximately 55% has adeleterious effect of retaining the micro-particles through a sugarcrystalline structure that bonds the elements to their supportingsurface. As previously noted, the stabilizing reagent consists of highconcentrations of sugars and proprietary compounds. We have found thatwhen the water component is allowed to evaporate, thick residue of thissugary compound is left behind, which under low humidity conditions hasa tendency to crystallize and become a fairly rigid structure which cancause micro-particles in this compound to become almost irreversiblyadhered to any surface with which it comes in contact and allowed todry.

It has been found maintaining relative humidity of a minimum of about55%, preferably about 60% or higher, softens (hydrates) the stabilizingcompound to the point at which the viscosity approaches that of water,and enables a pick and place process to proceed for placing themicro-particles in a microfluidic device. Under these conditions, onemay use automated tweezers or a vacuum picking head to grasp themicro-particles, e.g. withdrawing them from the capturing pockets or thesupporting surface of the pick-up plate, and placing them in their finaldestination, e.g. a microfluidic channel of a microfluidic device.

When ready to assemble, the pick and place technique elsewhere describedherein is employed in which the micro-particles are first disposed in agroove or pocket of a locator plate, from which they are picked bytweezer or vacuum tool.

An alternative, novel technique involves distributing themicro-particles in random fashion onto a flat surface not having groovesor alignment pockets. The advantage of this process is not requiring amicro machined plate component for the manufacturing process. Thedisadvantage is that the micro-particles are randomly distributed in apile. The tendency is for them to come to rest in a monolayer on thesurface, but with random orientation. Further, as a result of removingthe excess StabilCoat by centrifuge or wicking away the excessstabilizing solution, it has been observed that the micro-particles havea tendency to agglomerate into a dense monolayer concentration ofrandomly oriented elements. However we realize that there are solutionsto this problem. Individual micro-particles can be picked from thisdense concentration by use of an automated vacuum tip that engages thetop surface of the elements, such as previously described herein, incombination with a computer-control vision system used to identify anindividual element and its orientation from a detected image, and amotion system responsive to the vision system, which orients therelative relation of the vacuum pickup tip and the plate supporting themicro-particles in both X and Y coordinates and angular orientation.Similarly, automated tweezers may be operated with controlled motions inX and Y coordinates, and angular orientation, as described herein.

The GNRs immediately after immobilization are stabilized in a reagentthat is capable of protecting or stabilizing the catcher antibody on thesurface of the GNRs, e.g., StabilCoat®. GNRs are transferred to analignment plate and dispensed on to the alignment plate in a puddle withthe stabilizing reagent and the liquid form of the stabilizing reagentis used to aid in the assembly or the self-assembly of the GNRs intocertain alignment pockets. Because the stabilizing compound has a veryhigh sugar and salt content it is desirable to remove as much of themass of the liquid from the surface or around the surface of the GNRsprior to allowing any residual reagent to evaporate. As evaporationcauses a concentration of sugar content and salt content nearby acrystallization process occurs which acts to hold or cement the GNRsinto or onto whatever surface they are presented to. It is importanttherefore to remove as much of the residual reagent as possible and atechnique involving centrifugation was previously described wherein theexcess material is spun in a centrifuge and spun off of the plate andaway from the GNRs as shown in FIG. 9E. An alternative process that isconsiderably easier to implement a manufacturing process involves usingan absorbent pad placed at the edge of the channels associated with thepockets holding the GNRs wherein a capillary working process is used tosoak up residual StabilCoat® into the absorbent pad and draw out of thechannels through the channels and out of the channels and away from theGNRs.

Typical dimensions for the channels are shown in FIGS. 9A, 9B, 9C and9D. The plane view of the channels retaining the GNRs. The GNRs arelocated in pockets approximately 125 microns wide by 400 microns inlength separated from each other by a narrower channel approximately75-80 microns in width by 200 microns in length. The Figure is brokenaway, showing microtubes 1, 2 and n, where n may be as large as about50. The narrow channel is provided to aid in the flow of the excessreagent through the channels and away from the GNRs to the wicking padand narrower to prevent GNRs from migrating out of the retaining pocketthat they are intended to fall into. The length of the channels canrange typically from 5 millimeters to 25 millimeters depending on the oreven 75 millimeters depending on the size or the scale of themanufacturing process involved. The number of GNRs ranges from a fewhundred to a few thousand depending on the cross-section area. Perchannel, the number of GNRs is on the order of 50.

FIG. 2 is a side view of the GNRs on a surface or in a channel with apuddle StabilCoat® surrounding the channels and contained on the platearound the plate. It depicts a wicking pad in the process of being movedtoward the end of the channels full of the stabilizing solution. TheGNRs are completely submerged under the material.

FIG. 3 illustrates a wicking pad shown on the left top surface of theplate and having drawn the excess StabilCoat® away from the GNRs throughthe channels.

The micro-length tube elements while still in the plate can be furtherprocessed with a laser, preferably an ultraviolet laser, which could bean excimer laser, fluoride or krypton fluoride laser, with two beamsthat are spaced such that the ends and a an end margin portion orsection of the micro-length tube element are exposed perpendicular tothe element axis by a laser beam in a way that either ablates ordenatures the capture agent, e.g. antibody, from the ends of the elementas well as a section of the inside surface of the element. It is afeature of the laser configuration that the two laser beams areseparated by a fixed distance that defining the desirable width of theremaining band of capture antibody surface. The micro-length tubeelements within their pockets of the alignment plate can be allowed tomove back and forth with a degree of liberty, while still the laserprocesses substantially the ends of the elements and leaves a fixedwidth pattern near the center of the element, plus or minus a reasonablesome tolerance window.

It is possible instead to define a series of three or more laser beams,with gaps, such that the pattern produced by the various widths of laserbeams in the various gaps between the laser beams defines a pattern ofexposure in the tubular element that looks like and is useful as a barcode.

Further, it is realized as useful to have significant by-pass flow in achannel outside of the micro-flow element as well as through theelement. One advantage is simplicity of manufacture as the element canbe held but without being sealed and with no attempt to use cumbersomeadhesive to adhere the element to the channel walls. Another advantageis the avoidance of the risk of totally spoiling an assay because achance particle obstructs internal flow of one of the micro-flowelements when arranged in series in a liquid flow path. Havingsignificant by-pass flow on the outside, e.g. a flow greater than theflow through an element, thus “short circuiting” the element, ensuresthat despite one element being plugged or obstructed and flow stopped,the other elements will receive flow and the assay will only bepartially affected by the obstructing particle. It is realized furtherthat with concepts presented here, enabling the avoidance of havingactive capture agent on either the cylindrical exterior of themicro-length tube element or on its end faces, does not result in adepletion problem. The techniques previously described, of avoidingactive capture agent from adhering to the exterior cylindrical surfaceof the micro-length tube elements and laser treating the ends, thuscontribute to the practicality of employing the by-pass flow described.

Referring to FIG. 5, in general the advantage of so treating thesurfaces of the micro-length tube element, in reducing depletion, and inparticular, with regard to the considerations of by-pass flow, may beunderstood by considering the total surface area exposed to the coatingsolution as represented by the sum A+2B+C where A is the internalcylindrical surface, B is the end face, and C is the externalcylindrical surface area. It is pointed out that according to theconcepts articulated that it is possible to reduce the depletion area toA only, that being the only area that carries meaningful information.Indeed the reduction can be extended by suitably sizing the laser beamsto treat the margins of the inner cylindrical surface from which theactive agent has been removed or has been deactivated can be ofpreselected length. For instance, in a dilute solution, it may bedesirable to have more area than in a less dilute solution of theanalyte. Indeed, as previously mentioned, it is possible to have thelaser act upon capture agent in one or more mid regions of the area ofthe inner cylindrical surface, even form a code. Such patterns are notvisible when only in the state of an active capture agent, but madevisible or photo detectable by the captured fluorescently labeledanalyte.

As previously discussed, it is very important having no capture agent onthe outside surface of the micro tube elements, avoiding the potentialfor damaging the functional surface and negatively influencing theresults of the assay. As a result of transferring the micro-length tubeelements from an alignment plate to the microfluidic device or otherchannel it is quite possible for the outside surface to encountercontact with other hard surfaces, leading to damage of a functionalizedsurface. So it is beneficial to prevent binding of capture antibody tothe outside surface therefore, excluding signal that might arise fromthe capture agent coming from the outside surface. As an example of A,B, and C, where C is the exterior surface area of the micro-length tubeelement having a length dimension of approximately 250 microns, interiordiameter of 75 microns and an outside diameter of 125 microns, C beingthe exterior surface area is approximately 98,000 square microns. Bwhich is the two-times the end face surface area is approximately 15,600square microns, and A being the inside surface area approximately 58,000square microns. The total of A, 2B, and C is approximately 171,000square microns.

By cooperation of the processes, it is easy to eliminate the surfacearea associated with the exterior surface area of the micro-length tubeelement and the end face of the micro-length tube element, therebyreducing the total surface area by 66%. It is possible to further reducethe surface area of the capture antibody even on the interior surface toan arbitrarily small surface area. An example would be a narrow stripeon the inside of a 75 micron diameter tube, resulting in a total surfacearea of approximately 10,000 square microns. Total Surface Area:T=A+2*B+C. % Reduction of Surface Area without exteriorcoating=1−(A+2*B)/T; % Reduction of Surface Area without exterior andends coating=1−A/T; % Reduction of Surface Area without exterior andends coating=1−(A*0.5)/T. Example O.D.=125 microns, I.D.=75 microns,Length=250 microns. Without exterior: 57% Reduction of surface area;Without exterior and ends: 66% Reduction of surface area; Withoutexterior and ends and with 50% code: 83% reduction in surface area.

Assays run in the microfluidic device can use various types ofmicro-length tube elements with different capture agent present on theinside surface of the elements. For example, one type of micro-lengthtube element would contain a capture antibody associated with theantibody interleukin-6. Another one could be interleukin-2, and yet athird would be interleukin-12. Each of the micro-length tube elements ofrespective types can be placed into different channels and or locationswithin that channel, thereby determining at the time of performing theassay what type or what particular antibody was used in that particularlocation. That is a method for identifying the type micro-length tubeelement for what has bound onto that surface. In addition to usinglocation as a means for identifying a particular type of micro-lengthtube element, another means is using the striped code pattern created byselectively ablating or selectively denaturing antibody functionalityalong the length of the micro-length tube element for providing for acode, a bar code, which is then used to identify that particular type.

There are various possible ways of doing the ablation or creating theablated pattern, within the micro-length tube element using a laserbeam.

A wavelength in the range of about 193 nanometers to 250 nanometers ispresently preferred. One possible way is to establish a single laserbeam of a particular width and then translating micro-length tubeelement, then dispensing a fluence of laser to the element, turning thelaser off, translating to a new position then dispensing another amountof fluence thereby eliminating or denaturing only portions of aparticular tubular element. Another method, as mentioned previously, isto use an opaque mask to establish a particular pattern, and illuminatesimultaneously the entire micro-length tube area with the ultravioletlight. Yet another method is to scan the micro-length tube element usinga synchronized system of X, Y galvanometers.

Besides writing the code, one could scan simply the ends of themicro-length tube elements to ablate capture agent. It is anticipatedthat a feature size as small as 30-40 microns is possible with anultraviolet laser. For example, a 250-micron long micro-length tubeelement having a feature size of 30 microns would result in 8 possiblestripe zones and with 8 possible stripe zones, one could produce anumber of patterns. For example, a pattern using a binary coding systemwould lead to a total number of combinations of 28, which are 256possible combinations. The method:

Step (1): provide micro-length tube elements.

Step (2): apply coating, the manufacturing process, provide a coatingparticular of a capture antibody,

Step 3 a manufacturing process that coats only the inside surface of themicro-length tuber element. Two novel ways of achieving the this: One isproviding a capture agent, e.g. antibody, in a solution that contain themicro-length tube elements and agitating the liquid in an aggressivemanner, generating high shear force on the exterior of the elements,whereby binding occurs only on protected inside surface of themicro-length tube elements. The second means of coating only the insideof the micro-length tube element. The agitation process would preventcoating of the antibody on the outside surface only, but not the endfaces. So further reducing surface coating on the end faces wouldinvolve using a laser process. The second means of preventing coating onthe outside surface (but not chopped end faces) concerns themanufacturing process at the stage of drawing and coating endlessmicro-bore tubing, prior to chopping to form discrete micro-flowelements or micro-length tube elements. By adding a-bond-preventingcoating prior to the usual polymer coating prevents silanization of theexterior surface, preventing silane from adhering to the outsidesurface, and therefore defeating the ability for many capture agents,for instance antibodies and antigens, from adhering to the surface. Thiswould further prevent antibodies from being bound to the outsidesurface. In either case, the polymer coating is added to the glassfilament during its manufacturing process to maintain the intrinsicstrength of the glass filament. The process for providing ormanufacturing the micro-length tube elements, the raw elements without acoating, involve chopping the micro-bore tubing into the particularlength element, which for the preferred configuration is approximately250 microns, then removing the polymer coating using a sequence of acidand basic baths. Subsequent to the manufacturing process that coats onlythe inside surface of the micro-length tube element, the micro-lengthtube elements are then secured into a channel of a microfluidic device.

The channel of the microfluidic device is configured such that a portionof the flow is allowed to bypass the outside of the micro-length tubeelement. Micro-length tube elements are placed into a channel in a waythat, preferably, approximately two times the flow volume proceedsaround the outside of the tubular element as compared to the volumeproceeding through the inside area. So the ratio of the cross-sectionalareas of the by-pass flow versus the inside diameter is approximately2:1. Micro-length tube elements are then secured into the channelwhereby the channel wall is an elastomer, which allows the micro-lengthtube element to be placed in the channel, and the grippers used to placethe element into the channel can be released because the adhesion of theelastomer is sufficient to secure the element into the channel. Anyresidual adhesion between the micro-length tube element and the tweezersis overcome by the larger adhesive force between the micro-length tubeelement and the channel. Subsequent to that step, a top is then securedover the open channel containing the micro-length tube element. The topalso includes an elastomeric material. That elastomeric material is usedto compress and thus secure the element in the channel because the openchannel duct is of smaller depth than the outside diameter of thetubular element. The elastomeric “roof” or top thus provides a means ofsecuring the micro-length tube elements in their locations in thatchannel.

Referring to FIG. 8 et seq., another useful technique, with considerableadvantage, of completing the assembly between two pre-constructedsubassemblies, in contrast to using a non-permanent bonding process, isto form permanent bonds.

It is found that low pressure operation permitted by the generalorganization and design just described, can so-diminish the drivingpressure on the air to permeate the membrane, that the bubble problem islessened to the extent that an elastomeric membrane, such as PDMS, isemployable with significantly reduced risk of air penetration andbubbles than designs of the prior art. This has the advantages of lowcost and simplicity of manufacture, and enables achieving extremelyconsistent and sensitive assays.

A difference in the implementations now to be described is that themembrane or the flexible layer that is actuated by vacuum or pressure tooperate the valves and the pistons is made from elastomeric material anddifferent, advantageous techniques are used to fabricate the device.

One of the problems addressed concerns the surface area associated witha hollow flow element as has been depicted and as has been describedabove, i.e., an element having length less than 700 micron, preferablyless than 500 micron, and in many cases about 200 micron, and a borediameter between about 75+/−50 micron, that is fixed in a flow channeland exposed to flow of liquid sample. (Such hollow flow elements andassay devices based on them are available from CyVek, Inc., Wallingford,Conn., under the trademarks “Micro-Length Tube™, u-Tube™, and Mu-Tube™).Such devices are efficiently made of endlessly drawn micro-bore filamentsuch as used to form capillary tubes, but in this case, the filament isfinely chopped in length to form discrete, extremely short hollow flowelements, rather than capillary tubes. It is realized that capture agentimmobilized on the surface of such a device, applied by immersiontechniques, can raise a significant depletion problem. This occurs, forinstance, when attempting to characterize concentrations of an analyteat low levels such as a few pico-grams per milliliter, s is desired. Thephenomenon referred to as “depletion” occurs in which the concentrationof analyte in the sample being measured can be disadvantageouslydepleted volumetrically as a result of binding to a large active area ofthe flow element. This results in reduction of sensitivity of the assay,and therefore its usefulness. To explain further, any analyte in anELISA or sandwich type of amino assay on antigen will bind to a captureantibody in a way that is governed by a kinetic reaction, a dynamicprocess. While analyte such as an antigen binds to capture agent such asan antibody, the reverse also occurs, the bound analyte molecules unbindfrom the capture agent. The kinetics concern an “on” rate and an “off”rate analyte being captured and analyte being released. The capturereaction will continue, depleting the analyte in the ambient volume, andreducing its net rate of capture, until the system reaches equilibriumin which the rate of binding is equal to the rate of unbinding. Thegradual action occurs according to a substantially exponential curve.

The absolute value of the equilibrium condition depends on the originalconcentration of the analyte in the volume of sample being assayed.Increase in concentration results in a higher signal, decrease inconcentration results in a lower signal. In cases in which assaydepletion occurs, the concentration of the analyte in the sample isdetrimentally decreased over time. It is realized that hollow flowelements fixed in flow channel may present an excess of capture agent inthe volume of liquid sample to which the element is exposed, decreasingthe effective concentration of the analyte. The concentration decreasesat an excessive rate, relative to initial, starting point concentrationsought to be measured. While efforts to calibrate for this are helpful,such depletion ultimately lowers the sensitivity of the assay because,as the signal goes down, it approaches the noise level, and results in alower signal-to-noise ratio, i.e. an inherent reduction of effectivenessof the assay. (Already there are significant contributors to noise i.e.,background, nonspecific binding of capture antibody, fluorescence noise,electronic noise, etc.). Therefore, especially for detecting smallconcentrations, it is desired not to deplete the initial volume of theanalyte in manner that does not contribute positively to the assaymeasurement. Efficient ways to do that, as by somehow limiting theamount of exposed surface have not been apparent. This may be seen as aninherent problem with use of small detection elements of variousdescriptions that are coated by immersion or the like and used in animmunoassay or sandwich assay or even a molecular diagnostic type ofassay. One typically wishes to immerse the elements in capture agent,e.g. an antibody or some type of moiety that is a capture molecule forthe analyte to be sensed or detected, to uniformly coat all surfaces ofthe element. One object of invention is to overcome this problem withrespect to hollow flow elements characterized by an inside surface andan outside surface, or often also with two end surfaces. Adding up allsurface area over which a density of capture molecules is coated can addup to a surface area on the order of over 100,000 square microns. Thisis the case for a preferred form of hollow flow element formed ofsmall-bore filament, the element having on the order of about: a lengthof less than 700 micron, preferably about 500 micron or less, and inpresently preferred implementations, 200 microns. Likewise, the innerbore is found desirable to have within a range of 50 micron+/−25 micron,for achieving uniform coating by immersion and agitation. In onepreferred case, an element has an external diameter or width of 125microns, and an internal diameter or width of 70 microns. A particularproblem addressed here is to find practical approaches for accuratelyreducing active surface area of immersion-coated flow assay elements ingeneral, and in particular, hollow flow elements, and in particularelements of the dimensions mentioned.

A further problem being addressed here concerns treated hollow flowelements that are to be in fixed positions in channels for exposure toflow of sample. It is desirable to expose the elements in batch, in freestate to an immobilization process for applying the capture agent orantibody to the element surface, and then transfer each elementmechanically to its fixed position in a channel, for instance in achannel of a multiplex micro-fluidic “chip” (or “cassette”). It isdesired to use a quick and accurate placement process, for instance apick and place device mounted on an accurate X, Y stage. For suchpurpose, it is desirable to physically contact the tiny element forpicking it up from a surface and placing it in an open channel, which isthen closed to form a micro-fluidic passage. It is desirable to employgrippers, e.g. a tweezer instrument, or a vacuum pickup that contactsthe outer surface of the device. The pick and place action is madepossible by pre-aligning open channels to receive the hollow flowelements and the surface on which the free elements are supplied withthe automated pick-and-place instrument. This enables the grippers topick up and place the hollow flow elements precisely from supply pocketsto desired flow channel positions in which they are to be fixed. With avacuum pick up, it is possible to serve the hollow element in end to endabutting relationship in supply grooves, and engage the outercylindrical surface with the vacuum pick up. We recognize a problemarises with having an active capture agent, e.g. antibody, immobilizedon outer surfaces of an element. Such a coating is susceptible tomechanical damage as a result of the manipulation process. Outsidesurfaces of micro-flow elements come in contact with (a) a supplysurface, e.g. an aligning pocket or groove, (b) the transferringgrippers or vacuum pickup device, and (c) surfaces of the channel inwhich it is being deposited. All of these contact opportunities giverise to possible damage to the fragile coated capture agent, whichtypically is a very thin layer of antibody or the like adsorbed to thesurface of the flow element. This coating is often only a few moleculesthick, thickness of the order of nanometers or tens of nanometers, andis quite fragile. The net result of damaging a capture surface of theplaced hollow flow element is seen during read out of the assay. If thesurface has been scratched or perturbed in any way, that can give riseto an irregular concentration or presentation of captured analyte, thesignal can be irregular, and contribute to irreproducibility or poorperformance of the assay.

We thus realize it is desirable not to have immobilized active captureagent on the outside surface of a hollow detection element, andespecially the fine bore elements formed of micro-bore filaments, whereit is susceptible to damage, and where it contributes to increasing thetotal surface area of the capture agent or antibody that contributes todepletion.

The features described in the claims and hereafter address these andother important problems.

Discrete hollow flow elements are immersed in liquid containing captureagent, such as antibodies or antigens, and, after coating by the liquid,are picked, and placed into channels for flow-through assays. The hollowflow elements are in preferred form of discrete elements of length lessthan about 700 micron, and bore diameter of 70+/−50 micron, preferably50+/−25 micron. The flow elements are surface-treated so active captureagent, e.g. capture antibody, is not on the outside, or is of limitedoutside area. For this effect, the hollow flow elements are disposed ina bath of active agent and violently agitated, resulting in coating ofprotected inside surface, but due to extreme shear forces, a clean areaon the outside surface, for instance the entire outside cylindricalsurface of a round cross-section discrete element. In lieu of or inaddition to this shear procedure, a special filament-manufacturingprocess is conceived that results in preventing coating an exteriorsurface of flow elements with a predetermined capture agent. Captureagent on selected coated areas are ablated or deactivated with preciselypositioned laser beam, such as can be produced by a mask forsimultaneous treatment of a large number of elements, leaving residualactive agent of defined area on the inside surface of hollow flowelements. Residual capture agent, itself, on the inside of the elements,usefully defines a readable code related to the desired assay. Flowchannel shape is sized relative to flow elements fixed in the channel toallow (a) bypass channel flow along the exposed outside of a hollow flowelement to reach and flow through later elements in the channel in caseof clogging of the first element, along with (b) sample and assay liquidflow through the hollow flow element to expose the surface to captureagent and other assay liquids. Lacking the need to attempt to seal theoutside, the element can simply be gripped, as by an elastomeric sheetpressed against the element. Electrostatic attraction between flowelement and channel wall is employed to fix the element in position,overcoming any disturbing force of the placing instrument as it is drawnaway after delivery of the element. After assay, in the case of use ofepi-fluorescent detection, fluorescence is excited and read by specialscanning confined to the hollow flow element geometry. Locators areseeded in the recorded data, and used to locate the regions of interestin detected fluorescence data, e.g. from the elements. Code, writtenwith the capture agent substance inside the hollow element is readthrough a transparent wall of the element. A number of the features areor will be found to be useful with other hollow elements, for example,longer elements.

In respect of scanning, the purpose of this invention to deliver amethod for performing a fluorescence measurement of multiple immobilizedelements contained in a microfluidic chip. This method provides fordetermining the paths to be followed during the scanning, as well as theproper focus, and camera exposure. The method is based on a knowngeneral chip layout. The method provided results in the ability to placethe chip to be measured into the scanner and then start the scan withoutany additional manual settings required. The method does the rest, andproduces the desired fluorescence measurements as the results.

Certain aspects of invention involve eliminating or preventing theoccurrence of active capture agent on outside surfaces of the hollowflow elements, e.g. extended outside cylindrical surface, and/or endsurfaces, while leaving active capture agent on the inside surfaceunperturbed, or of a desirable area or pattern. Features addressing thisaspect include techniques to selectively limit the capture agent on theinterior surface and steps that act in combination with outside andinside surfaces to achieve the desired result.

For the specific advantage of reducing the overall capture surface area,two aspects of invention will be described, and the effect of theircombination. A first technique is employed to eliminate or preventcapture agent, e.g. antibody, from immobilizing to the outside surfaceof hollow flow elements. That is done during a batch coating process,and involves suspending discrete hollow elements in an Eppendorff tubeor other laboratory tube with the capture agent of interest andaggressively agitating fluid to impart disrupting shear forces to theexterior surface of the elements. Preferably this is achieved byvortexing the fluid at high speed, for instance employing an instrumentthat orbits the container at approximately 2000 rpm of the orbiter,about an orbital path with total lateral excursion of the supportingtable of the order about 0.5 cm, measured across the center of rotationof the orbiter.

The hollow flow elements are placed with a volume, e.g. a milliliter ofcapture agent, e.g. antibody. The appropriate vortexing speed isdependent e.g. on the nature of the suspension, e.g. the viscosity ofthe liquid chosen, and can be easily determined experimentally. It isset by observing whether the capture agent is effectively non-existenton the outside, long surface of the hollow flow elements, e.g. theoutside cylindrical surface in the case of the body being of circularcross-section.

The physical principle involved concerns shearing force on the outsidesurface of the element that acts to prevent binding of the capture agentto the surface through an adsorption process. One can observe whetherthe vigorous agitation is sufficient to shear off any capture agent,e.g. antibody that has already been bound to that surface. At the sametime, the inside surface is environmentally shielded from this shearingby virtue of the geometry which is tubular, and the micro-bore of thetube. This prevents vortexing from causing any turbulence to occurwithin the element. Only laminate flow conditions exist. With micro boreelements the Reynolds number is always low enough to ensure that thatlaminar flow condition exists on the inside surface. Under theseconditions, the velocity of fluid traversing in the hollow element atthe interior wall interface is by definition zero. So there is no shearforce involved there, whereas the outside is in a highly turbulent, highshear force environment. The shortness of the length of the elementsenables uniform coating of the inner surface, whereas longer elements,coated by immersion, are susceptible to detrimental non-uniform coating.

The observed result of aggressive agitation, e.g. vortexing, is thatfluorescence which is observed by performing a sandwich assay iscompletely absent from the outer cylindrical surface, or other shape ofa hollow element, whereas it is present in an observable way on theinside surface. In the case of square-end hollow flow elements,fluorescence is also present on the end faces of elements.

Vortexing is the presently preferred technique for producing the shearforces. The case showed here employs orbitally rotating the coatedelement in a very rapid manner back and forth in small circles at a rateof approximately a couple thousand rotations per minute, and anexcursion of about 25 mm.

However, any type of rapid oscillation that creates a high degree ofturbulence can be employed, so a back and forth motion, a circularrotation, anything that would very rapidly mix the fluids and createhigh shear forces will suffice.

In summary, hollow flow elements in the presence of aggressive agitationleads to removal of capture agent, e.g. antibodies, from outside surfaceof the elements, and prevention of their coating with the agent, butleaves the inside surface of the element in condition to immobilizecapture agent, e.g. capture antibodies, for subsequent interaction withanalyte of the sample.

As an alternative to the high shear technique, we conceive an alternateprocess in which, during the original drawing of the small bore tube,and prior to the point along the draw path that the usual removableprotective polymer coating is applied to the filament, that a non-stickcoating, e.g. sputtered gold, silver or graphite, is applied to thefilament, e.g. by passing through a sputtering chamber. Silane orsimilar coating must be applied to receiving surfaces before captureagent, e.g. antibodies will attach. However, due to the properties ofthe sputter coating, or the like, the surface will not receive thesilane or equivalent, then likewise, the active capture agent.

Another feature of invention concerns realizing the desirability andtechnique of removing coated capture agent from selected end surfaces ofthe flow elements and a margin portion or other portion of the interiorsurface. Preferably, following the aggressive agitation process, theelements are further processed using a laser elimination process thatremoves or de-activates capture agent, e.g. antibodies, from surfacefrom which the agent was not removed by the high shear process. Thosesurfaces include transverse end surfaces and a selected portion of theinside surface, leaving only an annular stripe on the inside surfacesized sufficient to process the assay, but small enough to reducedepletion of the analyte from the sample.

In a preferred form an ablating laser is arranged transversely to theaxis of elongation of the hollow elements with the effect that theenergy arrives though parallel to the end faces has a neutralizing orremoval effect on the capture agent that is on those end faces, as aresult of incidence of substantially parallel radiation, but also ofinternal reflection scattering of the radiation by the transparentsubstance that defines those end faces.

The net effect of two novel processes described, if used in novelcombination, is to leave only a band of selected dimension, which can besmall, of capture agent immobilized on the inside surface of the hollowelement. This can be done in a way that leaves one or more bandsseparated by a space of no capture agent. Thus one can generate a singleband in the center or a single band closer to one end or multiple bandsdistributed along the length of the element. These bands can be ofdifferent widths, can have different spacing, and can be of the form ofa code, e.g. a bar code, which is useful to encode the particular flowelement.

Further is a description of manufacturing techniques that have importantnovel features.

The short, hollow elements are first formed i.e. chopped, frompreviously supplied continuous small-bore filament into the short,discrete elements. They are then treated in batch manner.

A bulk of the hollow elements is then exposed in an Eppendorff tube towash buffer. After washing processing is performed, the buffer isremoved, and replaced with a silane. By use of this simple, low-costimmersion step, the silane is allowed to bind to all of the surfaces ofthe elements. Excess silane after a period of time is washed away withwater in a buffer. Then a capture agent, e.g. antibody, in solution isadded to the Eppendorff tube with the bulk of hollow elements andallowed to incubate overnight. The incubation is performed on theorbital vortexer for approximately 16 hours at 2000 rotations perminute. The order of 0.5-centimeter diametric displacement by theorbital motion. The orbital plate that contains the numerous Eppendorfftubes is approximately 6 inches in diameter, but the orbital motions isa circular pattern counterclockwise and then clockwise motion theorbiting causing the displacement of approximately 0.5 centimeters fromside to side, for instance.

After the vortexing process is completed, the net result is that thecapture agent has been immobilized on the inside surface of the hollowelement and also on the end faces but it is not present on the outsidecylindrical surface of the hollow element. The capture agent solution isremoved from the Eppendorff tube, which is replaced with a wash buffer,a wash buffer solution, and the wash buffer solution is then furtherreplaced with a stabilizing buffer, what we call a blocking buffer. Inthe preferred embodiment, a commercial material called StabilCoat®solution is used.

StabilCoat® blocking solution is introduced to the Eppendorff tube alongwith hollow flow elements, and then a portion of those elements isaspirated in a pipette along with some of the StabilCoat®, and dispensedonto an alignment plate. For tweezer pick up the alignment platecontains a series of rectangular shaped pockets, each designed toaccommodate and position a single element within a small space,preferably with clearance tolerance sized in microns, a space of 10 to50 microns between the element and the walls of the pocket. After theelements are allowed to roam on the plate, they fall into these pocketsstill in the presence of the buffer solution. The excess buffer solutionis removed from the alignment plate containing the elements by placingtheir plates with elements into a centrifuge or centrifuge holder andcentrifuging at approximately two thousand rpm, for 30 seconds, therebyremoving all excess StabilCoat® solution from the plate and theelements. This process is facilitated by the novel design of the plate,in which drain channels extend radially from the pockets.

(In the case of vacuum pickup continuous grooves are employed to receivethe treated elements, in greater density than enabled by the pockets, asthe elements can be close together, end to end, since pickup will be byengaging the cylindrical surface, not the end surfaces as is the casewith tweezers.)

The hollow elements while still in the plate are further processed witha laser, preferably an ultraviolet laser, which could be an excimerlaser, fluoride or krypton fluoride laser, with two beams that arespaced such that the ends and an end margin portion or section of theelement are exposed perpendicular to the element axis by a laser beam ina way that either ablates or denatures the capture agent, e.g. antibody,from the ends of the element as well as a section of the inside surfaceof the element. It is a feature of the laser configuration that the twolaser beams are separated by a fixed distance that defines the desirablewidth of the remaining band of capture antibody surface. The hollowelements within their pockets of the alignment plate can be allowed tomove back and forth with a degree of liberty, while still the laserprocesses substantially the ends of the elements and leaves a fixedwidth pattern near the center of the element, plus or minus a reasonabletolerance window.

It is possible instead to define a series of three or more laser beams,with gaps, such that the pattern produced by the various widths of laserbeams in the various gaps between the laser beams defines a pattern ofexposure in the hollow element that looks like and is useful as a barcode.

Further, it is realized as useful to have significant by-pass flow in achannel outside of the hollow element as well as through the element.One advantage is simplicity of manufacture as the element can be heldbut without being sealed and with no attempt to use cumbersome adhesiveto adhere the element to the channel walls. Another advantage is theavoidance of the risk of totally spoiling an assay because a chanceparticle obstructs internal flow of one of the hollow-flow elements whenarranged in series in a liquid flow path. Having significant by-passflow on the outside, at least as great as 50%, in many cases 75% orlarger, and in certain preferred instances 100% or more is highlyuseful. As least to some extent, this enables “short circuiting” theelement, ensuring that despite one element being plugged or obstructedand flow stopped, the other elements will receive flow and the assaywill only be partially affected by the obstructing particle. It isrealized further that with concepts presented here, enabling theavoidance of having active capture agent on the exterior, i.e. forcylindrical elements, on either the cylindrical exterior of the hollowelement or on its end faces, does not result in a depletion problem. Thetechniques previously described, of avoiding active capture agent fromadhering to the exterior cylindrical surface of the hollow elements andlaser treating the ends, thus contribute to the practicality ofemploying the by-pass flow described. Sizing of Hollow Flow Elements

It has long been accepted knowledge that the smaller the surface area ofthe capture agent, e.g. antibody, the more sensitive the assay is from atheoretical point of view. The desire has always therefore been to keepthe inside diameter of a hollow element as small as possible to minimizethat surface area. But it has now been determined empirically that,within limits, the performance of the assay is improved as that diameteris increased to an extent. It is believed this is a direct result ofnon-uniform coating by the batch process desired to be employed, as wellas probably some effects that occur during the assay in that it ispossible that there are perturbations in the amount of volume, totalvolume, that actually flows through the hollow elements in cases wherethe tube element diameter is small compared to an element of i.e. of 75microns. We have found that the internal diameter should be about75+/−50, and in preferred cases, 50+25.

It is preferable that the exterior diameter have a diameter or widthwithin the range of 1.2 and 4 times the internal diameter or width

For length of the hollow flow elements, best results are obtained withlengths of less than about 700 micron, and in many cases, less than 500micron. In a presently preferred form, the length is 250 um.

In some embodiments, it is preferable that the interior diameter have adiameter or width have a length to inner diameter of 20:1.

It has been discovered that the shorter hollow elements lead to greateruniformity of the coating of capture agent when coated by the batchprocess described herein, and as well, shorter hollow elements are foundto be more amenable to withstanding axial tweezing forces during pickand place motions.

As previously mentioned there are significant advantages in providingtwo subassemblies that are each fabricated on their respective solidsubstrates or carriers, which are dimensionally stable, thoughpermissibly flexible. The extremely small hollow flow elements (or otherdetection elements to be fixed in position within the cassette) areplaced into open locations on the mating face of one of thesubassemblies, prior to aligning. Once the subassemblies are aligned,the two subassemblies are brought together under bonding conditions toform one completed assembly, and fixing the embedded location of theelements. Then the two subassemblies are brought together to completethe fluidic channels. Bringing them together completes the valve andpiston devices as well as embedding the detection elements. Thesefeatures occur with the non-permanently bonded implementation.

Another implementation of the broad assembling concept will now bedescribed, employing permanent bonding features. We will refer now tothe Figures beginning with FIG. 27. The following is a list ofcomponents called out in FIG. 27 et seq.

-   20. Completed Cartridge-   22. Sample Inlet wells-   24. Buffer Inlet Wells-   26. Waste Well Reservoir-   28. Reservoir Well—Detect Antibody Reagent    -   Preferred Embodiment—Dried-   30. Microfluidic Channels-   32. Extremely Small Hollow Flow Elements (“Elements”)-   34. Microfluidic Valve Seats-   35. Microfluidic Valve Pneumatic Chamber-   36. Piston Fluidic Chamber-   37. Piston Pneumatic Chamber-   38. Elastomer Membrane-   39. Plasma Bonded Interface-   40. Arrows Depicting How-   41. Bypass Flow Path-   42. Glass Substrate-   43. Bulk Material-   44. Microfluidic Channel Walls-   46. Control Reservoir Layer-   48. Huidic Layer Sub Assembly—No Elements-   50. Huidic Layer Sub Assembly—With Elements-   52. Single Sample Four Analyte Microfluidic Network-   54. Microfluidic Valve—Full Assembly-   55. Piston—Full Assembly-   56. Reservoir/Control Plastic Member-   58. Pneumatic Interface Ports-   60. Piston Control Lines-   62. Valve Control Lines-   64. End of arm tooling (tweezer or vacuum probe)-   66. Pick and Place Arm (moves up and down)-   68. Source/Target X, Y table (moves in X and Y coordinates)-   70. Source of Hollow How Elements (groove or well plate)-   72. Target Microfluidic device-   74. End of arm tooling—vacuum-   76. End of arm tooling—tweezer-   78. Activated Surface

In FIG. 27, starting from the upper side, the subassembly 46, i.e. thecontrols/reservoir layer 46, is comprised of two elements, the upperinjection molded or machined plastic component 56 with a PDMS membranesheet bonded to its lower surface.

The bottom fluidic layer or subassembly 50 has detection elements, e.g.hollow short cylindrical flow elements 32. The fluidic subassemblyconsists of a thin glass sheet 42 with a PDMS gasket or sheetpermanently bonded face-wise to its upper surface, the sheet havingcut-outs defining fluidic channels between channel walls 44, the channelbottomed on the glass sheet 42, FIG. 28C. Before those two subassembliesare brought together, the detection elements are dispensed, in theembodiment shown, by pick and place action, into fixed positions in thechannels of the fluidic layer 48. The two subassemblies 46 and 50 arebrought together and bonded in a way that provides fluid-tight andleak-free operation, but also enables the actuation of valves andpistons by portions of membrane. One novel a feature of thisconstruction is that the two subassemblies as described, using a PDMSgasket, enables capture or embedding detection elements, here extremelyshort hollow flow elements, (Micro-Length Tube™ elements) into channels.Combining those two subassemblies into a single assembly provides thefunctionality of having microfluidic channels that contain the hollowflow elements as well as functioning valves and pistons. In afluidically robust and leak-free microfluidic structure, using theplasma-bonding process, known per se, to perform the numerous functionsdescribed, securing the detection elements in place and forming thevalves and pump diaphragms in a way that completely seals the channels,together with a process to be described that defeats plasma bonding atthe exposed valve seat contacted by the PDMS membrane.

The fluidic subassembly is assembled by covalently bonding PDMS toglass, and then upper assembly, the reservoir assembly is formed bycovalently bonding PDMS to plastic. The dominant advantage is theplacing the discrete, small detection elements, the hollow flowelements, into open channels prior to assembling.

The importance of the technique also relates to enabling theimmobilization of capture agent, e.g. antibody, onto a solid substratein an efficient batch process, thereby allowing many thousands of theseelements to be fabricated in one very simple batch process, which iscost effective and highly reproducible. The process itself is absent ofprocess parameters that would cause damage to biological content, andcan be a room temperature process.

Thus features of the concept include bringing together subassemblies tocapture elements in a fixed position, the capture (or detection)elements having been pre-prepared in batch process, with the finalassembly, which employing a bonding process, especially the permanentplasma bonding process to join the subassemblies, and doing it in aselective way at the valve seats by repeatedly locally deflecting andbringing in contact the valving surfaces, which will now be described.

Valve Break-in Process

-   -   Connect pneumatic control input ports to externally controlled        pneumatic line/s    -   Actuate all valves using vacuum (5-14 psi) to draw membrane up        into pneumatic valve chambers.    -   Bring surface-activated (e.g. plasma activated)        Reservoir/Control layer into conformal contact with Fluidic        Layer.    -   Momentarily apply pressure (1-10 psi) to valve control lines to        force PDMS membrane into intimate contact with the PDMS surface        of the Fluidic layer. Allow contact for approximately 1-3        seconds before switching back to vacuum pressure in control        lines.    -   Perform initial break-in of valves by rapid performing a        sequence of actuations between vacuum and pressure for        approximately 20 cycles, over a time period of 1-2 minutes.    -   Continue to cycle valves with vacuum and pressure over a period        of 5-20 minutes, depending on the surface activation and thermal        history of the PDMS surfaces. Once the initial break-in cycles        are performed, a slower and more protracted actuation sequence        is preferably used to prevent the slow inexorable bonding of the        PDMS surfaces, until all inclination for bonding is prevented,        which can be achieved by actuating the valve with pressure for        up to 1 minute followed by intermittent actuations with vacuum        so as to break any newly formed bonds. Continuing this process        for up to 20 minutes has been shown to completely prevent future        permanent bonding between the valve membrane and the valve seat.    -   Other materials which have molecular bonding capabilities when        like surfaces are bought together may also be employed, and the        molecular bonds destroyed at valve seats in similar manner,        Description of Valve Break-in Process

Native PDMS, comprised mainly of repeating groups of —O—Si(CH3)2- ishydrophobic in nature, and, without special treatment, has a tendency toadhere to, but not permanently bond to other like surfaces such as PDMS,glass and silicon. However, upon treatment with oxygen plasma or thelike the methyl groups (CH3) are replaced with silanol groups (SiOH),thus forming a high surface energy, hydrophilic surface capable ofbonding irreversibly with other like surfaces containing high densitiesof silanol groups. This irreversible bonding process occurs viacondensation reaction between OH groups on each surface resulting incovalent Si—O—Si bonds with the concomitant liberation of water (H2O).

Oxygen plasma and similar techniques have control parameters such aspressure, power, and time all of which determine the concentration ofsurface OH groups. Higher concentrations of OH groups lead to morecovalent bonds between the two surface and therefore higher mechanicalbonds. Left exposed to atmosphere after oxygen plasma or similartreatment, the hydrophilic surface will undergo “recovery” back to itsnative hydrophobic state via migration of short, mobile polymer chainsfrom the bulk to the surface. Full “recovery” occurs over a period ofhours at room temperature and can be accelerated with increasedtemperature and retarded by storage in vacuum and/or low temperatures.This is accommodated by storing activated substrates at −50 C in vacuumbags for several days to lock-in the hydrophilic surface treatment priorto bonding.

Since the bonding mechanism follows a fairly slow condensation reaction,which involves the liberation of, water over a period of several minutesto a few hours before completely consuming the available OH sites, it ispossible to interrupt this process before completion. Once completed,the bond strength between the interfaces is comparable to the bulk tearstrength leading to an irreversible attachment of the two materials.Attempts to separate the layers at this stage will lead to bulk damageof one or both of the layers. However, interruption of the bondingprocess by mechanically separating the surfaces during the early stagesof the bonding cycle is found to irreparably damage only the smallnumber of formed bonds between the two surfaces. The tear strength ofthe bulk is considerably higher than the interface bond, thereforeseparation produces no irreparable damage to the bulk. Also, if thebonding process is interrupted early enough (typically in first fewseconds), then the force required to separate the layers is little morethan the adhesion force required to separate untreated layers. Bringingthe layers back into contact for a short duration (typically a few moreseconds), will initiate, and interrupt bonding again. Each time thiscycle is repeated, potential bonds are incrementally eliminated untilall such bond sites are consumed and the material reverts back to havingthe properties of the untreated material.

In a preferred novel technique, microvalves are formed between layers ofPDMS by surface activating, e.g. plasma activating, the PDMS or similarsurfaces, bringing them into contact and then activating the valves toopen and close in such a manner that permanently disrupts bondingbetween the flexible membrane and the valve seat, but results incomplete and robust bonding elsewhere over broad surfaces to hold thedevice together.

Device Manufacture

Referring to FIG. 27, a product employing the concepts described is aconsumable microfluidic cartridge for the purpose of quantifyingantibody concentrations in human plasma samples. The cartridge, such asshown in FIG. 27, contains on board provisions for sample inlets, inother words, a reservoir that will receive a sample to be analyzed, e.g.a blood plasma or serum sample.

A completed cartridge 20 contains sample inlet wells 22 for receivingpatient plasma or serum sample or other type of bodily fluid, includingcerebral spinal fluid, or urine. It will also contain a buffer inletwell 24, buffer being a reagent used during the processing of the assay,a waste reservoir well 26 designed to contain all of the reagents andsample that flow through the microfluidic channels and that are nolonger needed all self-contained on the microfluidic cartridge, alsocontaining a reservoir well 28 which has contained in it a detectionantibody with a fluorescent label. The preferred embodiment, thedetection antibody will be dried down in the channel or in the reservoirand rehydrated during operation using the buffered contained in bufferwell 24.

Referring now to FIGS. 28A, 28B and 28C, FIG. 28A shows the microfluidicchannels containing 4 independent microfluidic channel groups containingthe extremely small hollow fluidic flow elements, referred to hereafteras elements. FIG. 28A shows those four channel groups each containingsix channels 30. There are extremely small hollow flow elements 32,microfluidic valve seats 34, and pistons 36. The extremely small hollowflow elements are formed in a batch process with a capture antibodyprovided on the inside surface of the elements and those elements areplaced into channels 30.

Example of dimensions of the hollow elements: The length of thepreferred embodiment is approximately 250 microns, the inner diameterapproximately 75 microns, and an outer diameter of approximately 125microns. FIG. 28B is a blown up schematic of the hollow elements shownin two parallel example channels.

In presently preferred practice, the channels are wider than theelements, and the elements are attracted by near electrostatic force toadhere to one channel wall, defining by-pass flow paths on the otherside.

FIG. 31 shows a cross-sectional view of a hollow flow element in channel30 with space surrounding hollow element on the outside of the element.FIG. 31 depicts hollow element 32 in microfluidic channel 30 with flowarrows 40 depicted, the hollow element as captured by the top surfaceelastomer membrane 38 and on the bottom surface by glass substrateelement 42.

Typical dimensions for the glass substrate layer 42 are 200 micronsthick of boro-silicate glass and the elastomer membrane layer element 38has typical thickness of 100-200 microns. Also providing the channelsare an elastomer, PDMS material typical 100-150 microns tall thusforming the microfluidic channel. Also shown in FIG. 31 the elastomermembrane layer continues both to the left and to the right as well asthe glass substrate continuing to the left and to the right and oneither side containing one or more parallel microfluidic channels alsocontaining hollow glass elements, glass layer element 42 is bonded toelastomer wall, a micro-fluidic channel wall 44, previously formed in asubassembly process using a covalent bonding technique involving plasmaactivation of the PDMS surface and subsequent contacting and thereforebonding to the glass layer, the hollow element is inserted into thatchannel.

There are additional channels 30 in parallel. The purpose of parallelchannels is to isolate different antibodies from each other forpreventing cross-reactivity.

Channel depth is less the diameter of hollow element that are picked andplaced against one of channel walls such that electrostatic forcesbetween the element and channel walls release the placing device, e.g.tweezers or vacuum pickup, from the element. In this process, by movingin an “L” shaped motion, laterally at the end, increases theelectrostatic attraction and allows the tweezer to be released fromengagement with the element and tweezers to be removed. Channel 30enclosed by bringing into contact both ends of elastic membrane 38 ofcontrol/reservoir 46. Elements are retained in channel 30 betweenelastomeric 38 and glass 42.

FIG. 29 shows schematically two example channels containing a series offour spaced apart elements 32 and by-pass flow space 41.

FIG. 21 is a top view of the fluidic layer sub-assembly 48 with elements32 in channels 30. The assembly 50 contains the elements.

In FIG. 21 four sets of microfluidic single sample, i.e., four analytenetworks 52 are shown, each network is designed to perform an assay withits own respective sample.

FIG. 30 is a blowup schematic of a single channel 30 containing fourelements 32 and microfluidic piston chamber 36, and valve 54 having seat34, FIG. 30A.

FIG. 30 depicts by arrowheads, flow through the bypass flow path 41around the hollow element 32 as well as through the element.

Referring to FIG. 21, the channels 30 are formed by glass substrate andmicrofluid channel walls formed by knife cutting sheet of PDMS of110-micron thickness.

FIG. 27 shows forming the fluidic area 48 by bringing together glasssheet 42 and the unique cut-patterned PDMS sheet 44 using knowntechniques.

Reservoir/control plastic member 56 (containing fluidic reservoirs forsample, 22, assay buffer 24 and reagent waste 26) is bonded to PDMSmembrane 38 to form control/reservoir layer 46.

FIG. 21 is a top view of the fluidic layer sub-assembly with elements 32in channels 30. The assembly 50 contains the elements.

In FIG. 21 four sets of microfluidic single sample, i.e., four analytenetworks 52 are shown, each network is designed to perform an assay withits own respective sample.

Referring to FIG. 21, the channels 30 are formed by glass substrate 42and microfluid channel walls formed by knife cutting sheet of PDMS of110-micron thickness 32

Referring to perspective of FIG. 22, layer 56 and membrane 38 are readyto be assembled by plasma-activated molecular bonding. FIG. 26 is a topview depicting final assembly. Pneumatic interface ports 58 are adaptedto match with computer-controlled pneumatic control lines that providepressure and vacuum actuation to valves 54 (formed by membrane 38 andmicrofluidic valve seat 34) and pistons 55 (the pistons being formed byelastomer membrane 38 lying over piston fluidic chamber 36 and pistonpneumatic chamber) piston control lines 60 and valve control lines 62.The piston pump formed by membrane 38 sandwiched between 37 and 36 isactivated by vacuum in one direction and pressure in the other.

For pneumatically controlled microfluidic systems, there is need forforming a fine, closely-spaced pneumatic channel network with highfidelity over an extensive area. One important use of such networks isfor actuating a distribution of microfluidic valves or systems ofmicrofluidic pistons and valves that constitute microfluidic pumps. Asis well known to those in the microfluidic field, there are significantproblems associated with doing this economically and reliably. Theproblems become more acute as the extensiveness, complexity and greaterminiaturization of the microfluidic network increases. Many of theproblems relate to materials, material handling and manufacturingtechniques.

In the case of using high precision molds to form three-sidedmicro-pneumatic channels integral with a body that may perform otherfunctions, there is extreme cost and inflexibility involved in creatingthe precision molds and in making mold changes required over the life ofthe mold. For many molded parts, there can be difficulty in achievingsufficiently intimate bonding with another material to close openchannels and achieve air-tightness under both positive and negative airpressures.

In the case of forming a composite channel with walls of one materialand top and bottom closures of different materials, it is difficult tofind the right materials and manufacturing techniques that meet allneeds. Form instability as well as difficulty in achieving intimate,airtight, long-lasting bonds under positive and negative air pressureare among the problems. During the production of such a device, whenformed of parts that need to be joined to complete the fine features ofthe pneumatic system, there is a particular need for the finely formedparts to have dimensional stability in handing to achieve registrationand proper mating with cooperating parts. Without precise registration,desired pneumatic pressure levels may not reach microfluidic valves orpistons to operate them in proper sequence.

These considerations apply especially to microfluidic assay devices thatperform multiplex assays, e.g. ELISA, in which in one device, multipleassays of bodily fluids are performed. In many cases, for instance indrug development, it is desired that multiple isolated samples besimultaneously subject to multiple assays.

There is particular need to find a combination of materials andmanufacturing techniques that meet all of the many requirements ofmicro-pneumatic structures described here and otherwise well known inthe field of microfluidics.

As a specific example, it is desired to provide a highly quantitativeimmunoassay device capable of receiving multiple samples, for instanceapproximately sixteen to sixty four samples on a single portablemicrofluidic cartridge, and provide, for each sample, a microfluidicnetwork capable of quantitative determination of a number of analytessimultaneously, for instance conduct four to eight different assays, theassay cartridge having macro sized features for containing patientsample (e.g. blood serum, urine), reagents such as buffers and secondaryantibodies, and waste.

Especially, it is desired to find an efficient approach to manufacturingsuch a portable microfluidic assay device of overall (“footprint”)dimensions of, e.g., 5 inches by 3 inches. In such device it isimportant to have the microfluidic channels closely packed with otherchannels and features, of a distance of the order of four to eight timesthe width of a pneumatic micro-channel, with, for instance, channelsizes on the order of 100 microns or less, for instance, channel widthsapproximately 100-150 microns and depths of 100-150 microns, withprecision in features of 10-20 microns of tolerance. In many cases,macro-size features such as sample wells and reagent reservoirs aredesired to be incorporated in the portable device, typically of severalmillimeters in dimension, i.e. several millimeters cross-wise andseveral millimeters deep.

To be of practical utility, it is necessary to find a way to provide theentire assembly reliably and inexpensively despite the optimum qualitiesof the materials for various features being different, and the need tojoin them in a reliable manner while retaining full functionality.

The invention is especially useful in microfluidic assay devices whichhave a pneumatic channel component, a fluidic channel component, and aflexible membrane joining the two. For this type of construction, aswell as more generally, we have solved the foregoing problems, inparticular with respect to the pneumatic component of such system, asfollows.

We found an excellent starting material for forming the pneumaticchannels comprises a double sided pressure sensitive adhesive sheethaving a non-fluorescent central layer formed of rigid material,non-fluorescent adhesive on each side, and peelable liner layersprotecting the adhesive and we found a process for forming the pneumaticchannels and features by processing this sheet by using a CO2 laser,followed by very simple bonding process. The laser is used to ablatepneumatic micro-channels and other structures by cutting entirelythrough the core, the adhesive layers and the liners to form thesidewalls of the desired channels and other features. Thus a pneumaticchannel has a side wall formed partly of the inner core layer of theadhesive sheet, and partly by the adhesive itself, at both sides of thecore layer. This technique is found to form fine precise features havingsizes on the order of 100 microns or less, with channel widthsapproximately 100-150 microns, and depths of 100-150 microns, withprecision in features of 10-20 microns of tolerance.

The dual-sided pressure sensitive adhesive sheet with suitable lowfluorescence is available commercially with size up to 27 inches, forinstance. It is important that the material have very low tendency tofluoresce when exposed to an excitation laser such as green laser or redlaser used to excite fluorescence in the conduct of epi-fluorescentreading of fluorescent-tagged analyte at the capture sites of an assay.Mylar™ (polyester), a material often used in the manufacturing ofpressure sensitive adhesive sheets as a structural component upon whichthe adhesive is applied, has a high degree of auto-fluorescence whichinterferes with the process of taking a measurement in the cartridge,and is inappropriate for use with assay cartridges intended to be readyby an epi-fluorescence or other stimulated fluorescent emission process.

It has been found that a core layer of polypropylene is excellent forthis purpose. A polypropylene layer of approximately 2 mils (50 micron)thickness with the adhesive on each side of 1.8 mils or 45 micronsthickness is found to perform very well with silicone adhesive layers. Asuitable product is sold by Adhesive Research under the productdesignation AR 90880 having layers of silicon based adhesive, known asSR26 silicon adhesive.

An example of a product formed by the techniques described is a portableconsumable immunoassay cartridge (cassette) constructed of severallayers, the pressure sensitive adhesive with channels formed bythrough-laser cutting being one of the layers integrated into thecartridge. That layer, with its peel strip removed, is attached to thebottom flat surface of a rigid reservoir layer that defines the macrofeatures previously mentioned, i.e. sample wells and buffer and reagentreservoirs. Laminated to the bottom surface of the pneumatic channellayer by the second pressure sensitive adhesive sheet, with peel stripremoves, is a membrane layer which is formed on a 100 micron thick PDMSmembrane containing fluidic vias that are aligned with vias in thereservoir layer and the adhesive sheet layer. This assembly is bonded tothe fluidic layer, elsewhere described, that bonding being effective tocapture discrete detection elements that have been introduced to themicro-fluidic channels and connect the microfluidic channels of thecartridge, though the vias, to the sample wells and reservoirs elsewheredescribed.

The rigid reservoir cartridge layer is either a machined plastic bodyapproximately 6 to 14 millimeters thick or an injected molded plasticbody approximately 6 to 14 millimeters thick having reservoirmacro-features located on its top surface, the features approximately 3to 6 millimeters in dimensions, and with fluidic vias from thereservoirs penetrating through the bottom of the reservoir layer,aligned with vias laser-cut in the pressure sensitive adhesive layer.The adhesive sheet with laser-through-cut pneumatic channels, vias andother features is laminated to the bottom surface of the rigid reservoirlayer in alignment with the vias in the reservoir layer so that thefluidic vias are arranged to transport sample and reagents from thereservoir layer to the fluidic layer through the vias in the reservoirlayer through vias in the pressure sensitive pneumatic layer and vias inthe following membrane layer. To then enter into the fluidic layer Alsocontained in the pneumatic layer are features associated with valves andpistons in addition to the fluidic vias. These features can be ovalsapproximately 800 um long and 500 um wide in the case of valves, or 3000um long and 800 wide in the case of pistons with long channelsconnecting these features along the entire surface of the substrate andterminating at the pneumatic actuation ports located at the end of thedevice in a series of pneumatic vias.

One of the challenges in creating a highly functional disposableimmunoassay cartridge is in constructing one that has a large number offeatures which therefore provides a high degree of functionality. Forexample, one which is capable of running multiple samples preferably 16to 48 different samples and for each sample the ability to preciselyquantify several analytes, 4-8, on one cartridge. Such requirementsoften drive the complexity and the need for a high density of fluidicfeatures, including valves, vias and piston pumps.

For each sample there is on the cartridge an independent fluidic circuithaving the ability to perform measurements of up to 8 unique analytes.The independent fluidic circuit is fluidically isolated from all otherfluidic circuits on the cartridge and is used to perform the samemeasurements in parallel on different samples. The current designsqueezes as many as 20-30 different fluidic features, such as valves,piston pumps and vias into an area of approximately 200 squaremillimeters (10×20 mm), as well as the pneumatic channels that connectthe features.

A cartridge that measures 16 individual samples would have 16independent fluidic circuits. However the functionality of each circuitis identical which means that every circuit is architecturallyidentical. So across the cartridge there would be 16 buffer inlet valves(one for each circuit) as well as 16 valve banks associated with each ofthe detect reagents, waste outlets and pistons. Since the circuits areidentical copies it's possible to share pneumatic control lines acrossall circuits and use a small set of independently controlled pneumaticchannels, limiting the complexity of the instrument that runs thecartridge. So for example, a cartridge with 16 samples running up to 8different analytes, could for example have as few as 7 pneumaticchannels where each of those pneumatic channels connects the same set offunctional features located in each of the independent fluidic circuits.The functional sets would include banks of valves for example a bank ofvalves that allow the detect reagents to flow at a particular time or abank of valves designed to close off and isolate a set of fluidicchannels from one another in the manifold region of the fluidic circuit,or a bank of valves located at the output or a bank of pistons. Sets offunctionality are connected to each other through a single contiguouspneumatic channel which terminates at one end at the pneumatic interfaceand the other end at the last feature in the string of connectedfeatures. Pneumatic channels in an effort to intersect with the sets ofactive features at every circuit are required to serpentine back andforth across a microfluidic cartridge, never overlapping one another, inan effort to cover all of the features located on the surface of thecartridge. Long continguous channels, as long as 10 to 20 inches inlength. And also as a result of the high density of pneumatic channelslocated on the devices it is necessary to keep channels, pneumaticchannels as tightly packed as possible in order to accommodate the highdegree of functionality required to run such an assay. As a result ofthese long channels tightly packed and located on a cartridge, andhaving a serpentine like path nature it was discovered that lasercutting a PSA based film for the purposes of creating these pneumaticchannels had the deleterious effect of being structurally unsound.During the manufacturing process or immediately following the lasercutting process it was discovered that the substrate with such formedchannels was unable to structurally support itself and retain therequired necessary dimensional tolerances.

As a result, features intended to have very tight physical tolerancesfor the purposes of forming the precision actuation of valve and pumppiston were lost as a result of having this physical instability due tothe formation of the long serpentine like channels back and forth acrossthe substrate.

The solution to the problem involved interrupting the channel formation,so rather than forming long contiguous uninterrupted channels thechannel were broken into segments approximately 20 to 30 millimeters inlength, and depending on the nature of the channel path if it involvedturning a corner for example or channels were closely packed to oneanother then the segment lengths could vary from 10 millimeters to 30millimeters. These channel interruptions were formed by making thechannel path with short, un-cut gaps approximately 150 microns inlength, forming bridges between the channel segments approximately 150micron. The result is a structure that is entirely self-supporting,which can be handled without the concern of losing the registration orthe intended tight dimensional tolerances. The channels now havinginterruptions in them and not having the continuity of air flow from thepneumatic input to the final terminal structure at the end of thechannel as a result of the bridges is made functional again by deployingshunts either directly underneath the bridges in the membrane layer ordirectly above the bridges in the reservoir layer, as shown in FIGS. 56Iand 56J.

In the case of shunts being formed in the membrane layer shown in FIG.56I a hole or a via is cut in the membrane similar to those used for thefluidic vias. In the case of the shunt being formed in the reservoirlayer as shown in FIG. 56J a small pocket is machined into the bottomsurface of the reservoir layer or it can be formed in the process ofinjection molding a piece of plastic involving the formation of thereservoir layer.

Once combined, the channel network of pneumatic channels and the shuntswhose alignments overlap with the bridges form a contiguous pneumaticchannel capable of actuating all of the features, such as pistons andvalves, located throughout the area of the cartridge. Such channels havecross sectional dimensions of approximately 150 microns by 150 microns.

The benefits of the approach include lower cost of manufacturing andhigher precision in feature locations. Because the raw material for thepressure sensitive adhesive is relatively inexpensive per cartridge(<$1/cartridge) and because the relatively high speed of manufacturingthese channels also results in a relatively low cost yet high precisionstructure necessary to implement the precision actuated pistons andvalves in a pneumatically actuated microfluidic device.

The overall cost impact as compared to injection molding or machining orother methods used for forming similarly such pneumatic channels issignificant.

Although one could injection mold such a piece, there are high costs andlong development times associated with developing an injection moldingprocess as the inflexibility of an injection molding process to adapt tochanging design concepts. It is anticipated that a number of variationsor configurations of cartridges will be supplied resulting in the needfor some flexibility in the formation of the pneumatic circuits, andwith injection molding each component requires a unique mold. Whereaswith respect to using the present invention, the same equipment, thelaser set up can be used with a different program to execute drawingsthat have been made.

One of the other important benefits is, the implementation of themembrane layer, which is an integral part of the fluidic cartridge as itis responsible for a number of functions. Its responsible for closingoff the channels and making them closed fluidic channels, for containingelements placed into such channels for the flexibility associated withforming microvalves and pistons. A PDMS membrane is necessary as anintegral part of the microfluidic cartridge and needs to be permanentlyadhered to the pneumatic channel surface whatever that pneumatic channelsurface is formed in. In the case of an injection molded plastic, thebonding process between the PDMS membrane and the PDMS is difficult andcostly as it involves multiple steps typically, and also is limited to asmall subset of plastics such as polystyrene, polycellphone, COC andCOP. Some of those plastics are unsuitable for the formation of areservoir layer that is formed in a thick such as 6-12 millimeter thickhaving both macro features on one side and micro features on the otherside. In the case of COC and COP, the cost of such plastics makes theformation of a device such as that prohibitively expensive. Which thenrelegates one to a very few number of available plastics such aspolystyrene.

Although some plastics such as polystyrene make good candidates for sucha process, most others, such as polycarbonate and PMMA are not goodcandidates. So developing a cartridge around a more flexible machineprocess then transferring it to injection molding would be difficultbecause the material choices aren't well suited for both processes.

Typically one starts with a machining process to wring out the designand then transfers it to injection molding once the design has beenstabilized and locked down essentially. Which is really a directconsequence of the high nonrecurring expenses associated with injectionmolding.

But you're bringing working towards the fact that the material forinstance with pressure adhesive on both sides can be easily bonded toany

Exactly, that is sort of the point, is that the pressure sensitiveadhesive because it is naturally adherent or naturally adheres to nearlyany clean smooth surface, the available range of plastics and processingprocesses are opened up widely. One can then employ polycarbonate orpolystyrene or any number of plastics without regard to the bondabilityof the PDMS membrane to that surface of plastic.

And just as an aside, if you were to make the pneumatic layer as a partof injection molded or machined part, what is done to make it bondableto a PDMS membrane?

Well, there's a process that involves plasma activating the surface ofthat plastic surface and then exposing it to an intermediate bondablelayer such as a silane or an organosilane type component which readilyadheres to both the plastic surface and the PDMS membrane surface.

The examples of FIGS. 43A, 43B, 44A and 45A illustrate PDMS bonding toPDMS. For a region R₁ in which bonding of the bondable surfaces is notdesired, FIG. 44B shows deflection of the two PDMS layers in oppositedirections by elastic deformation, and dashed lines indicate the layersin region R₁ relaxed, un-deflected. Adjacent regions R₂ of the layersare retained in contact, either solely by initial surface-to-surfacebonding, or with the added benefit of outside confinement orcompression, indicated by arrows P. Thus the Figure illustrates the twoconditions achieved cyclically during exercise of the make and breakprotocol previously described, enabling bonding at contiguous regions,but bond prevention in a selected region of the potentially bondablesurfaces.

For instance, for ease of tooling and product design, instead ofemploying physical engagement and mechanical movement to produce themake and break conditions at region R₁, it is frequently desirable thatthe make and break contacts be caused by application of fluid pressuredifferential evenly across the region R₁ of each layer. (The term“fluid” is here employed in its generic sense, to cover all “fluids”,i.e. liquids or gases). Use of fluid pressure assures even loading andprevention of disrupting distortion of a layer during the cyclical makeand break protocol. To achieve the deflected condition, a deflectioncavity at the backside of region R₁ provides space for the deflection.The adjacent regions R₂ of the layers are retained in contact, eithersolely by surface-to surface bonding, or with the added benefit ofconfinement or compression.

For reasons of convenience and economics in many situations, it is founddesirable to employ differential gas pressure to produce thedeflections. For instance, thus can be avoided the need for design ofspecial mechanical moving devices or the need for drying associated withthe use of liquid pressure. The gas pressure differential can beachieved by application of positive gas pressure to the interstitalspace, as by a special channel, with the benefit of being able to usehigh values of pressure to speed the operation or for use where bondsare formed rapidly or are of high strength.

In many cases however, it is found desirable that gas pressuredifferential be produced by application of vacuum to the outside of alayer to produce its deflection. One advantage is that the manufacturercan employ channels and cavities of the device itself, such as thoseassociated with pneumatic operation of the device during its normal use.By this the manufacturer can reduce the need for special, costly toolingand extra manufacturing steps. An example is the manufacture ofmicrofluidic cartridge device described herein.

Referring to FIG. 44B, in the case of applying vacuum on the backside(outside surface) of a PDMS layer to produce gas pressure differential,the cavity is a closed vacuum chamber engaged upon the backside of thePDMS layer. FIG. 44B illustrates such a deflection chamber on each sideof the pair of contacting membranes surfaces. The vacuum-actuateddeflected state of region R₁ is shown in solid lines while the dottedlines illustrate the natural relaxed and undeflected state when there isno vacuum is applied to the vacuum chambers. Preferably, indeed,positive pressure is applied to both chambers, having the effect ofenhancing the momentary contact, and therefore lessening the time neededbefore another break phase of the cyclical is performed, thus speedingthe neutralization of the layers in regions R₁.

FIG. 45B illustrates a deflection chamber on only one side of theassembly, deflecting a single membrane. The opposing membrane is shownrigidly backed against a planar surface to which it may previously havebeen adhered, to ensure that it remain stations when ron R₁ pulls away.

FIG. 43C illustrates a single deflection chamber and an opposing valveseat on planar surface in construction similar otherwise to that of FIG.45B.

In all the cases of FIGS. 44B, 45B and 43C, the deflection chamber maybe formed by manufacturing tooling constructed only for that purpose andthen removed. In other cases as has been shown in the examples of themicrofluidic assay cartridge shown in this application, the deflectionchamber is in fact part of the final microfluidic product, with numerousobvious advantages with respect to tooling cost and economy ofmanufacturing steps.

FIG. 56K diagrammatically illustrates a microfluidic cartridge having acomplex microfluidic (liquid) channel network, a large number ofmicrofluidic valves formed by the make and break protocol, and otherpneumatically controlled features that are all actuated simultaneouslywith the valves.

There are many obvious technological advantages and beneficial effectsof perform this make and break operation simultaneously on manydifferent valves, or on many other features, such as those describedbelow, or on combinations of many valves and many other features. Forinstance, from a manufacturing point of view there can be great easewith which a complicated structure can be made by applying only a simplepneumatic force in the form of a vacuum or pressure and actuating all offeatures at once. This of course includes the benefits of low cost andlow complexity of the manufacturing process.

As has been indicated, there are numerous advantages to using cycles ofpneumatic vacuum and positive pressure applied on the exterior or thenonbonding side of the flexible sheet. While such vacuum deflection ofthe make and break protocol in manufacture of portable microfluidicassay cartridges has great advantages, more broadly viewed the essenceof the activating fluid aspects of the invention has to do withdeflection of a region of a membrane layer by application ofdifferential fluid pressure across the layer no matter what medium isemployed or on which side the greater pressure is applied. Inparticular, there are important circumstances in which advantage isobtained in creating differential gas pressure across the layer bypneumatic forces applied between the sheet-form layers, i.e. applyingpositive pressure to cause the deflection of one or both layers andnegative pressure between the sheet-form layers to cause collapse andcontact.

One of the advantages of employing positive pressure relates to the factthat in employing vacuum to deflect the membrane, the maximum pressurethat can be applied is 1 atmosphere, approximately −14 psi, which limitsthe deflection forces applied to a membrane whereas a positive pressureapplied between the sheets is nearly unlimited in magnitude to causeseparation to occur between the sheets.

In respect of applying positive pressure to produce the outward membranedeflection, two alternatives for the deflection cavity will bedescribed, that of a deflection slot defined by walls that engage theouter surface of the layer, but that is open and un-limiting withrespect to the deflection distance for the layer, and that of a chamberthat also has the walls, but is closed with a ceiling. The walls of anopen or closed cavity for pressurized deflection define the physicalperimeter of the area of the deflection, thus defining regions R₁ andR₂, and hence the area over which the make or break process occurs. Inthe case of use of a simple open slot, the limit of deflection is afunction of the elastic properties of the membrane and the pressureapplied, and may be most useful in the use of moderate positivepressures, or where there is ample margin for error regarding thestrength of the membrane. The use chamber which, in addition, has aceiling creates an additional physical limit for outward deflection,which can potentially protect the membrane from deleterious effects oftearing or bursting the membrane, useful e.g. where particularly thinmembranes or particularly high outward forces are to be employed.

In the discussion so far, the flexible, deflectable sheet has beenmonolithic, and in the examples of FIGS. 43A, 43B, 43C, 44A, 44B, 45A,45B and 56K, it has been PDMS. A composite sheet may be used instead,comprised of multiple layers, the basic requirements being that theoverall sheet is flexible to be capable of elastic deflection, and thatthe inner surface be bondable. While not necessary according to broadaspects of the invention, it is advantageous that the bondable surfaceof the composite still be surface-activated PDMS, either pre-formed as aseparate sheet or as a coating on a carrier sheet which itself may bemonolithic or a composite. In the example of FIG. 46A the compositecomprises a bondable pre-formed layer joined to a layer of anothersubstance. For instance the bondable layer may be surface-activated PDMSand the back layer of a pre-formed layer of a material other than PDMS,for example a sheet of PDMS laminated to a second pre-formed flexiblesheet of a compatible substance, or one that can be rendered compatibleby use of a treatment, such as flame or plasma treatment or by use of anintervening layer that can be bonded to both. For instance the secondlayer may be a pre-formed sheet of Mylar™ (PET), Polycarbonate orPolyurethane produced as blown or cast film sheet, as appropriate forthe resi and the circumstances. FIG. 46B illustrates a flexiblepre-formed back sheet coated with a thin coating of PDMS materialtypically ranging from approximately one to three microns in thickness,having its surface activated in preparation for a bonding process. Theback layer for instance may be Mylar™ (PET) of thickness approximately 5to 15 microns.

In still another implementation, not shown, an essentially monolithiclayer of PDMS may carry an exterior coating of another substance, forpurposes such as improving the gas-barrier properties of the composite.

FIG. 46C illustrates a laminate structure similar to that of FIG. 46B,but with a flexibility-increasing feature. It is useful for thecondition in which a flexible outer sheet bonded to PDMS sheet (or itcould be a flexible sheet having a PDMS surface coating) wherein theflexible sheet is less flexible than PDMS and it is desired that thecomposite exhibit increased flexibility, e.g. to increase the deflectioncapability and thus the flow capacity of a valve or pump formed by thedeflectable membrane. The principle being illustrated is use of aninterruption or reduction in thickness of the back layer. Theinterruption can be a single moat in the back layer, extending aroundthe perimeter of the defined area R₁, or, as shown, a series ofconcentric moats that allow a greater displacement to be generated as aresult of allowing the flexibility of the PDMS to perform the majorityof the stretching during an activation of either a vacuum deployed orpressure deployed activation protocol.

By way of summarizing certain technological advantages and beneficialeffects of features regarding materials selection for the flexiblemembrane:

PDMS has the advantages of being a low cost material that is easilybonded, flexible and easily machined or otherwise easily formed intochannels surfaces to cooperate with the deflectable membrane featuresdescribed. In the specific case of laminated structures, including thatof FIG. 46C is the ability of a material other than PDMS to block airpassage or reduce the overall gas permeability coefficient of thestructure. Whereas PDMS is known to have a high degree of gaspermeability other plastic material such as polyester, polycarbonate andpolyethylene exhibit extremely low permeability relative to PDMS. Thiscan be of great benefit in microfluidic type devices in which positivegas pressures are used for actuating valves, preventing gas permeationthrough the membrane into the fluidic channels thereby creating bubblesthat can have a deleterious effect. For instance, under many assay flowconditions, air bubbles that enter the reagent stream can attach to thecapture agent and prevent binding; air bubbles can prevent completewetting of surfaces, and thus inhibit the capture agent from capturingan antigen of interest; and air bubbles can also displace fluid in themicrofluidic channels causing the microfluidic system to become lessstiff from a fluidic point of view and also increasing the variabilityof flow rate, producing uncertainty of flow rat that can impairquantification assays.

With specific reference to the technological advantages and beneficialeffects of the last Figure, FIG. 46C. It employs a series of narrowlydefined moats or channels that are cut into the more rigid yet somewhatflexible air-impermeable backing sheet part of the flexible sheet/PDMSlaminate. The moats allow the stiffer component, e.g. Mylar™ (polyester)or polyethylene to deflect using the underlying flexible PDMS to act asan expansible spring, therefore achieving greater deflection. Thus,decreased permeability of the relatively stiff backing layer, todecrease air permeability within a microfluidic valve can be obtained,while achieving enhanced membrane deflection away from a valve seat toallow adequate or improved flow across the cooperating valve seat whenthe membrane is deflected to open position. This is a novel feature inits own right, as such benefits can be obtained even when differentapproach is employed to join materials of the device.

Referring again to FIG. 45B, the single deflectable membraneimplementation, a fixed bondable surface opposite to a flexible membranePDMS bondable face is not limited to the same material, and there arecircumstances in which advantages are obtained by using a differentbondable surface. Referring to FIG. 47A, the deflectable membrane has aPDMS bondable surface, but the opposite bondable surface is provided bya rigid silicon based material including crystalline or amorphoussilicon, amorphous silica, silicates and ceramics. Benefits of differentthermal conductivity, electrical conductivity, or the ability to addelectrical contacts, as in the case of silicon or having the beneficialproperties of silica in the form of optical clarity low autofluorescenceoptical smoothness or the special hardness properties and insulatingproperties of ceramics can be of advantage.

FIG. 47B illustrates the accommodation of a flexible membrane with asurface-activated PDMS bondable surface to a synthetic resin or metalbased device employing an intermediate bifunctional layer. For examplesurfaces of well-known plastics including COC (cyclical olefin polymer),COP (cyclical olefin copolymer), polycarbonate, polysulfone, polystyrenecan be surface-activated or metals such as aluminum or iron that eitherreadily form oxide layers can be employed. Such surfaces can be modifiedwith an intermediate bifunctional layer such as an organol, to create anoxide layer.

In a preferred implementation, a portable microfluidic cartridge 2 isplaced into an operating and scanning instrument by the user. It entersin a receptacle or reception area 6 at which the cartridge is retainedfor conducting the assay while scanning.

A suitable receptacle is shown in FIGS. 57 and 57A, and the relationshipof the receptacle and cassette when in assay/reading position is shownin FIG. 57B and the detail of FIG. 57C. An implementation of the overallsystem is shown in exploded view, FIG. 58. FIG. 58 includes x, yprecisely movable stage 13 that moves the cartridge on its carrierrelative to the stationary objective lens.

Referring to FIGS. 57 and 57A, cartridge 2 and the cartridge receptacle6 are shown with a clamping mechanism 12 and pneumatic interface 8. Aseries of computer-operated solenoid valves 9 that move on the stagewith the cassette apply positive and negative air pressure to ports thatinterface with the positioned cassette.

FIG. 59C illustrates a microfluidic configuration within a cartridge,illustrating both a series of fluidic networks and a pneumatic channelnetwork to actuate on-board membrane micro-valves and micro-pistons inthe fluidic network. The fluidic network in FIG. 59A comprises eightdiscrete microfluidic circuits closed by an over-lying elastic membrane,e.g., a continuous layer of PDMS. Each of those circuits has a number ofmicrofluidic channels, valve locations, and piston locations. Portionsof this membrane are located at formations in the channel that definevalve and pump cavities. The corresponding portions of the membranedefine movable elements of the valves and the piston.

The pneumatic channel network FIG. 59B is shown as an overlay in FIG.59C. It matches the fluidic network with respect to the various featuresthat need to be actuated.

FIG. 60 is a magnified view of one of the circuits of FIG. 59C,illustrating a number of the micro-features including valves, pistons,and the various reagent or reservoir inputs including the sample, thebuffer (wash), the assay reading dyes, the secondary antibodies and thewaste. The four elements GNR shown in black in each of the fourindividual (isolatable) channels represent glass nano-reactors (GNRs)embedded in those channels. This illustrates the basic micro fluidicunit replicated a number of times in the cartridge depending on thenumber of samples that the cartridge is designed to accommodate. Theassay protocol flow sequence shown at the left of FIG. 60 starts withthe prime flow step, and is followed by sample step, wash step,secondary antibody step, another wash step, a dye step for reacting toattach reading dye to the captured moiety, and finally another washstep. This illustrates an example assay sequence capable of beingperformed in this microfluidic structure. Each one of the fluids:sample, secondary (e.g., secondary antibody), wash, and reactive dye iscaused to flow from its respective inlet well by activation of the pumpsformed by each piston and upstream and downstream valves, with the endresult of captured moieties at the detection elements that are labeledwith the reactive dye, ready for reading to quantify the result of theassay. Examples of volumes employed on this device include the sample at20 microliters, a buffer of 150 microliters (as shown in the table)—thetotal volume of the microfluidic circuit is approximately 1.8microliters.

In FIG. 98, four micro channels on a portable microfluidic cartridge areillustrated, each having two monitor positions. The further discussionrelates to the first set of monitor positions 1, 2, 3, 4 in respectivechannels. In FIG. 98A, three different operations of an illustrativeassay with discrete phases are represented by times t1, t2, and t3. Inphase 1 at time t1 at four different locations on a cartridge to samplefour channels, the tracer signal is detected to be at the expectednominal value within the acceptance rate. It is therefore considered asuccessful phase 1 disposition. Phase 2, at time t2 the nominal level isnear zero, which might indicate that a buffer or some fluid thatintentionally had no tracer was properly flowing in the channels at thatparticular phase. In phase 3, at time t3 third reagent or fluid in thechannel has a tracer level that is different from that of phase one butis detected to occur at its nominal value within its acceptable andexpected range. So the entire operation considering phases 1, 2, and 3would be considered successful. This represents proper operation with nofailures.

FIG. 98B illustrates another run of the same assay. In phase 1, time t1,a failure has occurred wherein the detected tracer signal occurs outside(here, below) the acceptance range. In channel 1, the tracer signal isshown present, but lower than the acceptance range, whereas in channel 4the detected tracer value is shown as not present. In FIG. 98C at timet1 of the assay run, all four channels are shown as having a detectabletracer signal below acceptable range. But note that all four signals areequal and uniform. This indicates that there is not an independentfailure mode within that cartridge but probably indicates that animproper dilution had been used to create the reagent that was used.

There are other failure modes such as the improper interface of thepneumatic seat which could lead to valves not opening fully or notclosing fully or pistons not operating in full extension or lift so thattheir fluid volumes might not be what was anticipated. A hierarchy ofsignal modes can be constructed, e.g., in the simplest case a signalversus no signal, a simple digital response, and in other cases wherethe quantitative value of the signal does not meet expectations.

In digital response, there is no quantification. The signal is eitherpresent or not. A further level of complexity involves quantifying thelevel and comparing that quantity to an acceptable level where there isrange of acceptable levels (acceptance range) not just on or off. Thatquantification technique might be used to determine whether a properdilution or proper concentration or proper reagent was used in theproper location. Another advantageous level of sophistication is inmonitoring and analyzing the signal structure over time, to obtain thetemporal response of the signal relative to an expected temporalresponse. That requires a more detailed explanation.

Referring to FIG. 99, the evolution of a detected tracer signal is shownwhile monitoring over time a fixed location in a microfluidic channel,for example a channel approximately 100 microns wide by 100 microns deepin a length section of approximately 20-50 microns long. Thus a veryspecific isolated location within the channel is monitored over threedifferent phases. The first phase shown depicts the condition of no flowoccurring within the channel but the channel has present in it a reagentlaced with a tracer of a certain concentration that provides a detectedsignal of any type, e.g., detected fluorescence. Phase 2 in FIG. 99follows the evolution of the signal, showing that it decreases over timeas the reagent with the tracer is displaced by a reagent without atracer, for example a buffer reagent or wash liquid that has no tracerin it.

The signal evolution decays very rapidly in phase 2 as the new reagentdisplaces the old reagent with tracer, and the signal goes down. Then inphase 3 of FIG. 99 the displacement process stops and the signal ismonitored with no net flow during phase 3. This represents successfulwashing of a channel.

The benefit of thus staring at one location is not only to watch thereal time evolution of what is occurring at the location but also in thecase of using a fluorescent dye that is photo bleachable, to look at thefine structure not shown in this trace but shown in subsequent tracesthat reflect details of exactly what is occurring in the microfluidicdevice.

Another benefit of staring at a specific location is to acquireinformation in the development of an optimized protocol. In thedevelopment phase for an ELISA or any other assay that involves multiplesequences of reagents and flushing followed by new reagents, it isimportant to know whether the displacement of the prior reagent iscomplete and how many cycles or how long or what type of flow rate forexample is required to ensure that the next phase of the assay processis firmly established. What is called “open protocol” refers to assaysthat are not monitored. For such assays, it is vitally important tocharacterize the microfluidic system beforehand. The technique beingdescribed may be used as a tool to be used during test runs of an assay,to characterize the system and optimize the protocol. So for example ifinsufficient flushing or wash steps were applied, then residual reagentwould be present at a phase that could be harmful to the performance ofthe assay. The presence of such errant reagent may be detected bypresence of its respective tracer. This therefore is an advantageoustechnique for evaluating performance and optimizing an assay protocol.

One implementation is to provide a cassette in development on an x, ymovable table. The table is indexed to any selected position relative tothe detection systems, e.g., optical system, and the assay can be runwhile detecting signal from that position. A commercial instrumentconstructed to run and read an assay has substantially all of thefunctionality required to generate development data that is fed backinto developing an optimized protocol.

FIG. 100 illustrates the time response of a tracer signal while staringat a single location in a microfluidic device, referring back to FIG.99, location 2, for example. The tracer dye present in the liquid in thechannel is selected to be photo bleachable progressively over time. Inan example, a red laser diode is employed to excite red-excitablefluorescent tracer material. When the fluid is stationary, a photobleaching process is observed, the detected signal decaying as afunction of time. The decay rate is dependent upon the laser power andtype of the dye and the concentration.

Flow may be introduced to the channel in an oscillatory fashion. Thepurpose of oscillating the flow in normal operation of an assay is toenhance the interaction of the analyte present in the unknownconcentrations sample with a capture moiety, e.g., an antigen in thesample with an immobilized capture antibody. A typical defined volume(“slug”) of liquid is used, of fixed volume that is much larger than thevolume exposed at the detection point. When portions of this oscillatingslug of liquid move away, this allows time for diffusion to take placein those portions to bring the material into equilibrium before it comesback for exposure to the capture site, and back and forth. Whenever itcomes back, the analyte in closest proximity to the capture moiety,e.g., some percentage of an antigen is captured and drawn out of thatsub-volume of the sample—depleting that sub-volume. Then as it is flowedaway, and diffusion allows that sub-volume to approximatelyre-equilibrate, to reach substantially equilibrium concentration—intime, it replenishes the sub-volume in the vicinity of the captureagent. And so the flow is oscillated back and forth to give maximumopportunity for all the sub-volumes to interact with the capture moiety,to substantially optimize the reaction by optimization condition withthe capture moiety.

Referring back to FIG. 100, the time response of a detected tracersignal is shown from a given monitoring location in a scenario withoscillatory flow. Fluorescent dye is used that is subject to photobleaching. One observes a peak-like nature, or an up and down signalstructure with peaks and valleys at a given time sequence andperiodicity, due to the oscillation frequency of the fluid back andforth. So there is no net flow of fluid away from the location, but withoscillation, there is an opportunity to replenish the photo-bleachedportions of the reagent with fresh reagent. In those cases where theflow rate is maximum, passing by the excitation beam quickly, the photobleaching decay rate is offset by replenishment of new reagent. That isshown in the peaks labeled “max flow rate.” At the turnaround pointswhere the flow rate of the oscillating flow goes to zero, the photobleaching decay is maximum that is indicated by the valleys, labeled“min flow rate” on this graph. From detail shown in this particulargraph, in addition to the frequency and the peak-like nature, one isable to see that there are two types of peaks, taller peaks and shorterpeaks. The taller peaks are associated with the flow that is beingdriven by the piston moving fastest, in this particular fluidicdevice—when the piston is under vacuum actuation. The smaller peaks aregenerated by the piston displacement when the piston is moving slower,being actuated a positive pressure. The negative actuating pressurevalue is greater than the positive pressure value and therefore inducesa greater rate of displacement of the piston. For example, the negativepressure for actuating the membrane diaphragm of the pump is about −8 or−10 psi, while the positive pressure actuation of the piston is underabout 4 psi.

The signature shown in FIG. 100 is indicative of normal operation. Inthe case where a pneumatic interface was improperly sealed or seated,then these peaks heights would occur at different levels, outside ofnormal acceptance range, so this is a type of failure mode that could bedetected. Another factor involved here is the flow rate, which dependsnot only on displacement volume of the piston, but also on the impedanceof the fluid in the microfluidic channel. If the impedance is increasedby the addition of blockage from a contamination source or some otherproblem, such as a detection element being misplaced in the channel,then the nature of the signature structure would be different from whatis expected and shown in this graph.

The trace shown in FIG. 100 and the detected values in FIGS. 98A, 98B,98C and 99, are acquired by capturing the fluorescence intensity duringsteps of the assay by an imaging system shown diagrammatically in FIG.23. It has an objective lens and a series of optical elements. Anexcitation beam from a laser is introduced to the monitoring location ata microfluidic channel, FIG. 102. The optics transform the stimulatedfluorescent object, see FIG. 102, to an image plane shown as a photodetector (but in a preferred implementation, a CCD camera). Theintensity of the pixels within a region of interest (ROI) captured onthe camera are summed (integrated) to produce a single intensity pointfor that frame for that moment in time.

In FIG. 102 the laser beam is shown in cross-section as an oval while arectangular box circumscribing that oval illustrates the region ofinterest (ROI) over which the pixel intensities are integrated toproduce a single resultant signal point. That value at this point intime is plotted as a point on the graph in FIG. 100.

The scanning system is adapted, during reading of assay results, tointerrogate a detection element on which assay capture agent isimmobilized, see the Scanning Figures described later herein. But inmonitoring mode, as depicted in FIG. 102, by relative movement betweenoptics and microfluidic system, the system is focused on a selectedmonitoring point on a fluid-carrying channel at a point in which thedetection element is not present.

The optical arrangement of FIGS. 101 and 102 is used to generate thesignal trace in FIG. 100 or the “snap shot” at a monitoring point inFIGS. 98A, 98B and 98C, or the measurements over time of FIG. 99.

Another important use of the novel tracer detection technique isidentifying the location of microfluidic channels with high fidelitywithin a portable cartridge (cassette), relative to a cartridge positionthat is subject to some variation in position on a scale relevant to thesmall features of the microfluidic cassette. This is described furtheron, after describing a preferred implementation of a scanning/assayconducting instrument.

Referring again to FIG. 102, for a suitable instantaneous image size, itis appropriate to use an excitation beam imaged through the objectivelens to a spot size of approximately 12 microns wide by 250 micronslong. The region of interest (ROI) of the camera that includes that spothas an area of approximately 35 microns width by 250 microns length. Themicrofluidic channel of the cassette that will be monitored has achannel width of approximately 180 microns.

Three scenarios for monitoring the tracer dye with such a beam are: (1)Continuous Scanning Modality. The microfluidic channels are scannede.g., with substantially constant velocity across all channels of amicrofluidic system, such as on a cartridge. In that case, the beamcrosses over the channels and measures the fluorescence intensity as afunction of position or as a function of time, as the channels arecrossed. (2) A Rapidly Shift-Momentary Dwell Modality. In this case thedetection axis is moved rapidly relative to the microfluidic system, toa selected location, followed by a momentary dwell, e.g., of a fewseconds, in which the scanning system stops, and the optical detectionsystem stares and collects tracer information or tracer signals as afunction of time at the fixed location. The sage then moves on toanother location to repeat the stare and this is done for a large numberof locations over the micro-fluidic system in a short period of time.(3) Long Stare Modality. In this case a single location is selected atwhich the system stops and starts for an extended period of time, e.g.,many seconds or even minutes. It can characterize a specific pumping orfluidic operational step. It is especially useful if there is suspicionabout that particular location, determined as a result of one of theprior two scanning modalities.

The system and method have aspects useful for monitoring duringoperation of every assay and others that are useful as a diagnostic toolfor design.

In respect of monitoring every assay, the Scanning Modality isespecially useful. It is used to scan across all channels of amicrofluidic system, e.g., on a cartridge, repeating this during eachphase of execution of the assay. It can also stop at various locationsfor a short period of time to collect a trace at that location, and stoplong term for staring to characterize the flow over time even in thecase of monitoring usual assay function.

For a presently preferred implementation of a microfluidic cassette andhow scanning can be accomplished, refer to FIGS. 59A, 59B, 59CFluidic/Pneumatic Architecture and FIG. 60 Fluidic Architecture andProtocol.

FIG. 61 illustrates the same microfluidic unit in a schematic fashion,showing four independent fluidically isolated microfluidic channels ineach of which the respective piston and valves define a pump dedicatedto that channel. The Figure illustrates controlled pneumatic controllines overlaid, terminating at a number of circles containing x's thatrepresent valves. Channels with fluorescent dye present produce highcontrast signal. Channel locations and chip orientation is preciselydetermined from scan profiles. Optimal focus is determined by sweepingz. The horizontal lines with arrows labeled “Find channel scan 1” and“Find channel scan 2” illustrate the path that a scanner or a stagecarrying the microfluidic system moves in order to identify thefluorescence intensity, and thus identify the location, of each of thefluidic channels. As the relative scan motion moves along that path, itwill produce a trace shown adjacent to the schematic. The upper andlower black and white traces shown in the center illustrate thefluorescence intensity being high in channels 1, 2, 3, and 4 based onthe peaks shown on the traces. This illustrates that the scanner whilescanning across that path encounters high fluorescence because of benigntracer in liquid in each of the channels. The locations of the channelsare then determined very precisely by taking the encoder informationsuperimposed on that trace. The precise location of those channels isthus determined relative to the absolute coordinate frame of thescanning system. It is possible now to produce high resolution andhighly aligned scans because the precise locations of the scans are nowdetermined with respect to the x, y stage. Thus, while a holding systemfirmly fixes the position of cassette within the operating scanningposition, its exact location at the level of micron accuracy need not beprecise because of the trace-determined high accuracy detection. Anexample of a system that performs these operations is described below inrelation to the scanning Figures.

The primary benefit of the approach described, of precisely identifyingthe location of the channels, is to relax the requirement that thecartridge be precisely aligned on the stage by the user.

It has to be fixed relative to the stage but only within a fairly courseacceptance range. A benefit of the technique is that it does not requireprecise absolute (and costly) positioning by the user. It permits thepresently preferred clamping implementation about to be described.

The apparatus for conducting the assay and simultaneously scanning forbenign tracer presence will now be described with reference to FIGS. 57,57A, 57B, 57C and 58. In FIG. 57, assay cartridge (cassette) 2 is shownabove carrier plate 4, in preparation for being placed into receptaclearea 6. When placed, the cartridge will make intimate contact withpneumatic interface 8, so that pneumatic controls (solenoid valves 9)can actuate appropriately to apply air pressure and vacuum via interface8, to actuate the micro-valves and pistons on board the cassette andthus perform the assay. The cartridge is retained in the receptacleinterface 6 by retaining clamp 12. In FIG. 57B, clamp 12 is shown withthe cartridge in place in receptacle area 6. In FIG. 57A, the retainingclamp is shown in the process of being closed. FIGS. 57B and 57C, andthe exploded view of FIG. 58 illustrate the relationship between thecarrier plate 4 and cartridge 2 in it and the rest of the mechanicalassembly of the instrument in an exploded view. The precision x, y stage12, chassis 16, heat plate 14, and optic subassembly 18 are shown. Notshown is an enclosure for the system that excludes ambient light formthe cartridge or other microfluidic assay system, and from the opticalsystem, such that ambient light does not interference with fluorescentexcitation and detection during performance of the assay and during thereading of assay results.

Referring especially to FIG. 57C, a further magnified view, thepneumatic interface and the clamping pneumatic interface 8 are shownwith the cartridge 2 in intimate contact with pneumatic interface 8while the clamping anvil 26 is resiliently compressed, providing a forcecompressing the cartridge against the pneumatic interface. The clamp isheld in its down position by a latch.

Previously described in earlier provisional application regarding thefinding of the channels algorithm (as Scanning FIGS. 79, 80 and 81,below), we have FIGS. 62, 63 and 64. FIG. 62 again illustrates fourisolation channels and the path of the scanning sweep performed toidentify the precise location of each of the channels. FIG. 63 shows atrace obtained by such a scan using white light illumination as opposedto using fluorescence with benign tracer, by laser-basedepi-fluorescence process. The signal shown in FIG. 63 illustrates a highlevel of signal followed by fairly small dropouts or spikes illustratingwhere the edge of the channel or shadow is formed as a result of whitelight illumination as it impinges upon a channel. One can see in thetrace that the signal change is a fairly small percentage of the overallbackground signal, the signal dropping from approximately 3500 counts tojust under 2300 counts. This low signal makes the signal processingpotentially challenging in that small aberrations or perturbations ofthe signal caused by other means could interfere with trueidentification of the channel. FIG. 64 shows a magnified view of two ofthose spikes illustrating both the left and right edge of a channel asthe scanner proceeds across the channel. The description or the presentinvention utilizing a benign tracer fluorescent dye provides for muchgreater signal to noise level for this particular trace, and thereforeis a significant improvement that can be substituted in the system. Thesignal level outside of the channel where there is no fluorescent dyepresent is only the material use in the construction of the cartridge.The fluorescence of those materials is exceedingly low whereas thefluorescence found when crossing over a channel containing liquid withfluorescence dye is exceedingly high, giving a much higher signal tonoise level, and therefore greater accuracy and robustness.

In summary, a number of points of great value of the invention will bereviewed. An assay is performed under strictly controlled assayconditions, e.g., heated uniformly, and the controls (e.g., pneumaticvalves and pistons) are controlled precisely. This is done while some orall of the assembly is detected, e.g., translated in x, y coordinatesrelative to an optical axis of a detector (e.g., camera), such that thecassette is simultaneously detected, and tracer condition determinedwithin the various channels while the assay is run. In advantageousimplementations, the very same detection instrumentation is later usedto detect assay results from capture agent.

A simple technique to implement, having significant value, uses ascanner during the assay protocol to simply detect the presence orabsence of the tracer dyes at the various phases of performing the assayprotocol. The scanning process generates signal patterns that arecompared to predetermined anticipated levels associated with the normalperformance of the assay on the cartridge.

This may be readily performed by computer computations, or empirically,based on acceptance levels defined from a number of experimental runs todetermine normal levels are and some acceptable range of those levels.

This tracer-based process provides great value in determining whetherthe assay or cartridge did what it was intended to do during the assayrun.

The invention provides an entire system of monitoring methods that canbe employed in coordinated fashion to address the previously describedfailure modes, and others. For example, another failure mode notpreviously discussed is in the controls based in the bench top operatingand scanning instrument itself. If a control fails, e.g., a pneumaticpressure controlling solenoid valve, this failure is also detected. Thusthe invention provides a generic means of detecting a host of potentialfailure modes during a microfluidic assay system run and especiallydetermining whether a reagent is present or not in that channel, and ifit is the proper reagent in that channel.

Similarly, the invention enables simply scanning across channels withbenign fluorescent dye for the purpose of precisely locating themicrofluidic channels, for setting up scan parameters, e.g. for thepurpose of identifying optimal focus location, another important featureof significant value. This is in the set up process during the executionof the assay protocol that is described further within, in relation tothe Scanning Figures. While the cartridge is running the assay protocol,e.g., under pneumatic protocol, the scanner system can simultaneously beused to perform a number of measurements useful to the later detectionphase when the assay is completed. Those measurements include locatingthe channels based on the fluorescence properties of the channelscontaining liquid with the tracer dye. Also for determining the optimalfocus location—scanning of the z-axis or the focus axis to determine theoptimal location of the focus based on the fluorescence intensityprofile as well. These are for use in the final scanning performed afterthe fluidic phase of the assay to make the quantification.

The benefits of the concept primarily pertain to making a robustoperational system for making quantitative immunoassay measurements. Thereproducibility and the enhanced validity of the data quality providesvalue for this approach.

Process controls are routinely used in the art in measurements, forexample, in ELISA plates, controls are used as individual wells on anELISA plate. Researchers when running any type of instrument always wantto know or have positive verification that the measurement that theymade is believable, and it is performed the way it is expected to beperformed. The present invention is a means to producing that confidencein a microfluidic system and the data that the system produces.

Using the concepts herein, one may provide reliability scanners ormonitoring scanners, independent of any particular type of microfluidicsystem. Such scanners can be used by anyone running a microfluidic assayto monitor how it is performing.

But the invention is particular beneficial to a microfluidic cartridgein which a series of reagents are flowed in different volumes anddifferent timings, to positively identify that each one was performedproperly.

The fluorescent dyes used for benign tracers inherently should notinteract with the components of the assay. However if chemicalinteraction were found, it would be routine to chemically modify the dyeto make it more inert or more benign with respect to interferences inthe system. There are known conjugations that can be performed on thedye mark for such purpose.

The invention has special utility in respect of complex microfluidicbased systems that run a sequence of reagents, assays wherequantification is the primary outcome of the measurement

Many preferred embodiments leverage what is already available in asystem for making quantitative analytical measurement, e.g., existingsystems in which analytical measurements are being made over a number oflocations and therefore require a moving system to put the cartridge inthose locations or move the detector to those locations on themicrofluidic system, e.g. cartridge. Techniques following the inventioncan be readily retrofit into such systems. The invention is particularlyapplicable to situations in which the fluidic component and the opticalcomponents can perform simultaneously and the assay environmentalrequirements can be met. Temperature is an important parameter to becontrolled during the assay protocol. The temperature is typicallycontrolled for immunoassay systems at least to plus or minus one degreeand preferably plus or minus ½ degree C. In addition, absence of ambientlight or stray light is beneficial, so the assay is performed in a darkenvironment, as provided by an enclosure. The techniques of the presetinvention are compatible with these requirements.

Some of the techniques described have accuracy beyond what ordinaryblood laboratories can accomplish. For instance, the GNR's may be usefulin stationary, non-portable microfluidic systems, to make very accuratemeasurements. Techniques of monitoring described have applicability insuch instances, e.g. in high throughput blood testing.

This the inventors envision the invention applied to large, lessportable higher throughput machines having a high degree of accuracy ina diagnostic type of environment. The main point is that the diagnosticmeasurement must come with a certain degree of certainty in its accuracyand the novel method of enhancing the degree of certainty in the outcomeprovided here has wide utility.

A unique assay system will now be described, which involvespneumatically actuated valves and pistons for delivering precise volumesof reagents throughout a microfluidic disposable cartridge.

It is desired to have a bench top operating and reading instrument thatcan operate the assay cartridge to conduct an assay through timedoperation of pneumatic valves on the cartridge that cause flows offluids that have been placed on the cartridge prior to the assay, and tothereafter read the results.

The instrument needs to have significant robustness in terms of theuseful life of the instrument.

It is advantageous to provide the cartridge instrument interface on amovable stage capable of transporting the cartridge in X, Y scanningmotions relative to a fixed axis of the optical system that determineslocation of the cartridge and fluidic channels in it, and monitorsprogress of the assay and reads the results.

A key requirement is that the cartridge must interface with a pneumaticcontrol component on the instrument in reliable fashion so that nopneumatic leaks occur between the cartridge and the pneumatic actuationsystem.

The valves and pistons on the cartridge are controlled by pressure andvacuum provided by the instrument, and if a leak were to occur at theinterface between the cartridge and the reader, those valves would notactuate precisely and reliably. The result would be imprecise control ofthe flow of reagents on the cartridge and uncertain results with regardto the assay. This is because the assay depends upon precisely timedactuation and metering of reagents, precise volumes and precise timesfor exposure of the unknown sample to the capture agent, the subsequentflushing and washing of the sample prior to mixing, and exposure withsecondary capture agent and then followed by the subsequent washing ofthat component followed by exposure to a fluorescent dye, or regardingthe last two steps, alternatively, exposure to a fluorescently labeledsecondary agent.

The concept for reliably making that pneumatic interface involves acompliance component as part of the cartridge. In its preferred form, itis in the form of silicone rubber layer as part of the cartridge thatmates with a robust, rigid port component contained in the instrument.The rigid component contains a number of vias through which thepneumatic actuation is provided in the form of pressurized air orvacuum.

One of the important features is the novel arrangement by which both acompliant component and a rigid component are provided, that are broughttogether under force to form an airtight seal. The beneficialrelationship is that the compliant component is located on thedisposable element and not on the non-disposable side of the reader. Thebenefit is that the rigid component has a much longer life than acompliant component, as the rigid component would not undergodeformation over time, whereas a compliant component such as siliconerubber and other forms of rubber or even plastics would undergoinelastic deformation which eventually would lead to failure mode in theform of a pneumatic leak.

In the preferred implementation, the rigid component located on theoperating instrument is metal, either aluminum or steel, and thecompliant component is PDMS or silicone rubber carried by the assaycartridge.

The rubber is exposed and advantageously is provided as an extension ofone of the layers within the cartridge. It is provided on the bottomsurface of the cartridge, while the wells and reservoirs of thecartridge are provided on the top surface. The thickness of the siliconerubber is approximately 100 microns. In the preferred implementation itspans the entire surface area of the cartridge which could be 120millimeters by 85 millimeters, and its durometer is about 30 shore A. Aclamping system is provided to place pressure to bring the two togetherto form a seal that is maintained at numerous pneumatic vias. In theexample, there are seven vias positioned in close proximity. For examplethe spacing between vias is approximately 2 millimeters and the viadiameter is approximately one millimeter.

One of the important features of the pneumatic manifold interface isthat the rigid component on the instrument is constructed to have asmall cross-sectional area of contact. In this manner, only a smallforce is required to create significant physical pressure upon thecompliant material. The pressure locally compresses the compliantmaterial, thereby providing assurance of a leak-tight seal. The smallarea is achieved by providing a pneumatic manifold interface with verysmall dimensions in both the length and the width. In the example, theentire width of the interface seat is approximately 3 or 4 millimeterswide by 10 millimeters long, that includes all seven of the vias. Whenthe cartridge is in operating position on the operating/readinginstrument, its end with pneumatic interface vias rests on the rigidpneumatic interface part of the instrument, the other end of thecartridge being s supported by a fixed seat which holds the cartridge inlocation. X, Y direction constraint is provided by a set of four cornerretaining stands, these being tapered to enable easy insertion of thecartridge into the thus-formed receptacle pocket.

Pressure is applied to the cartridge only in one location to obtainstability in the Z coordinate. The force is applied downward through thecartridge in one embodiment by a roller connected to a leaf spring, theroller arranged to contact the top surface the cartridge and provide adownward force which compresses the compliant material on the cartridgeagainst the pneumatic manifold. In another implementation, as shownhere, a simple releasable clamp applies the pressure.

There are alternate techniques to achieve the clamping force as will beunderstood by a skilled person, including spring-loaded mechanisms,roller mechanisms, and motorized rack and pinion. A pneumatic solenoidor an electrical solenoid can similarly apply force to maintain theconnecting pressure.

Cost effectiveness and simplicity are usually the objective whendesigning the clamping mechanism. A motorized roller is a conveniencefrom a user point of view, but a swing bar as shown is simple,effective, and avoids potential failure modes.

The other significant feature on the movable stage is the pneumaticinterface manifold. The pneumatic control lines, the seven differentpneumatic control lines in this implementation, are controlled bysolenoid valves that are carried on the X, Y stage and connected viaflexible hose to a pneumatic manifold in pneumatic communication with avacuum pump and a pressure pump.

An important advantage of putting the pneumatic valves on the stage isthat only two flexible pneumatic lines and a flexible electrical controlcable are required to move during the motion process of the stage, sothat the pneumatic channels connecting the solenoid valves to thecartridge are hard fixed channels as part of the pneumatic interface.One of the advantages is reliability of using fixed machined channels ina robust metal or plastic component compared with flexible tubeconnectors that over time tend to fail. Another beneficial feature ofhaving the pneumatic interface as a fixed machined component mounted onthe stage is its contribution to the desired speed of the assay. Thisrelates to the ability to minimize the dead volumes of the pneumaticpassages. This is important in the operation of the cartridge becausethe valves and the pistons change states as a result of switching fromeither a vacuum state to a pressurized state. The speed at which thatoccurs is directly proportional to the dead volumes in those channels.For example, switching from pressure to vacuum requires the vacuum to becompletely drawn on whatever volume is contained downstream of thesolenoid valves. Using the features just described, all of thedownstream channels from the solenoid valves are extremely small, andthe distance between the solenoid valves and the chip is maintained in avery short distance. Yet another advantage of having the pneumaticinterface as a fixed machined component mounted on the stage having alow dead volume is in the ability to use low flow rate, and thereforeinexpensive vacuum and pressure pumps. Since the volume of the pneumaticlines downstream of the solenoid valves and the rate of states changesdetermine the average flow rate, it is desirable to keep the dead volumelow so as to allow the use of smaller, low flow rate pumps.

Speed is important because a large number of actuations must occurthroughout the assay protocol. There can be tens of thousands ofactuations and even tens of milliseconds or hundreds of millisecondsdifference can add up to a substantial loss of time.

There are two different active components on the cartridge, valves andpistons. The purpose of the valves is to determine which reagents flowand to which channels they flow. The pistons are the primary componentsfor motivating the fluid. They provide the positive and negativedisplacement to the reagents located on the cartridge, and so are theprimary elements for motivating fluid.

The features contained on the stage are the pneumatic interface with thesolenoid valves and cartridge and the clamping device. That is all thatis on the stage that moves. The cartridge on the movable stage isexposed to a fixed heater plate underneath, supported only 4 or 5millimeters below the surface of the cartridge and exposed face-to-facefor radiant heat transfer. The bottom surface of the cartridge where theactive capture elements are contained in the microfluidic channels mustbe maintained at a constant temperature between 35 and 37 degrees C. Theheater plate extends in the dimension that is actually slightly largerthan the surface area of the cartridge to cover its range of travel. Auniform temperature profile is thus maintained over the surface of thecartridge. The biggest advantage of that is that the temperature of thecartridge is easily maintained without having to control the temperatureof the entire reader enclosure. Thus there is no concern about heatingthe electronics and other sensitive components within the enclosure.Temperature control and stability is only provided at the criticalsurface.

Further, it is easier to control the temperature much more precisely inthis fashion, using radiant heat, than it is using a convective process.It also enables temperature stabilization to be achieved much quickerthan by a convective process. This is important for antibody kinetics.Antibody binding is temperature dependent so accurately controlling thattemperature is an improved way of controlling the rate of the kinetics.

This is particularly important because many of the assays desired to berun are “single point assays”. The assay is run for a fixed amount oftime and the resulting fluorescence signal is proportional to theconcentration of the analyte in the sample. Any other parameters thataffect the fluorescence needs to be controlled precisely so that thevariable is the concentration of the analyte. Temperature is asignificant parameter that affects the binding kinetics between theantigens and the capture antibodies. And so that needs to be controlledso that potential source of variation it is taken out of the equation.Also, a convenient feature for a user to be able to just turn theinstrument on and within a minute or two provide their cartridge andexecute an assay run.

One of the features of the instrument is to excite a fluorescence signalusing a laser and then capture that fluorescence with an objective lenswhile the stage is translated. A well-known epifluorescenceconfiguration is employed in which the excitation signal, provided by alaser or laser diode, is sent through an objective lens and then thereturning fluorescence is captured by the same objective lens and sentto an imaging CCD camera. The heater plate which held fixed directlyunderneath the cartridge is provided with a hole that allows both theexcitation and the emission signal to propagate through to the fixedoptical system.

All objective lenses have a so-called “working distance.” Key featuresof an objective lens include numerical aperture and magnification, whichwill determine the ability of the objective to capture the fluorescenceintensity in a very efficient manner and image that back to the CCDcamera. The working distance for typical 10× objectives is somewherebetween 5 and 12 millimeters. It is important to maintain a distance ofsomewhere between 5 and 12 millimeters between the objective and thebottom surface of the cartridge.

This critical distance is achieved despite the intervening presence ofthe heater plate. This is achieved by the heater plate being a thinaluminum plate, about one eighth of an inch thick with very thin heaterstrips, e.g., 1/32″ thick, adhesively attached to the aluminum heaterplate.

The total distance between the objective and bottom of the cartridge isapproximately 12 millimeters, the thickness of the plate is a smallfraction of that, it is 4-6, and the plate itself has a hole that allowsthe light to transmit through. The hole is sized such that the objectiveis brought up into the hole itself, fitting partially into the plate.

Sequence of Operation

The sequence begins with placing the cartridge onto the cartridgereceptacle pocket, then sealing that cartridge using a clampingmechanism, then after warm-up, actuating the pneumatic valves whichforces the reagents including the buffers and the samples and thedetection antibodies to flow in a very specific sequence for a specificperiod of time allowing incubation to occur. The incubation results inthe binding of the unknown antigen in the sample to the capture moietiescontained in the cartridge. The various reagents flow in a givensequence with intermediate wash steps followed finally by a fluorescencescanning process

In some cases, the stage remains stationery throughout the entireincubation process. In other cases, described herein, the stage is movedduring incubation to enable visual monitoring by use of tracers in thefluid. The final reagent process is to flow detection dye or fluorescentdye through the channels which then binds to the immobilized secondaryantibody. During this process, if not previously done using tracer dyes,it is necessary to move the stage because it is during this process thatthe fluorescence dye found in the channels is used as an identificationbeacon for identifying the location of the channels. The optical systemis actually used in coordination with motion control system to identifythe location of the channels by exciting with the laser the fluorescencein those channels and the locations of those channels are identified asa result of an increased signal where the channels come through.

The bench top unit implementation described here contains no liquids andno liquids flow between cartridge and the bench top unit.

In the following, we describe other novel details of a preferred benchtop unit.

Referring to the Figures, the bench top unit has just a few keysubsystems. The subsystem that holds the cartridge. The cartridge isplaced into a little receptacle area and located in that receptacle areais the pneumatic interface boss that has limited end surface area (“liparea”) for contact with the cartridge. It protrudes off of the surface,that is the highest surface. One end of the cartridge sits on that boss.The other end of the cartridge sits on a small rail on the other side ofthis containment area. These are corner guides that make it easier toplace the cartridge. A small arm contains on it a little spring loadedcontainment clamp. The spring loaded clamp bar comes down and rests onthe top surface of the cartridge, and pushes the cartridge down on tothe pneumatic boss.

On the opposite end is a lock and catch that holds the device in itsclamping position. The user pulls down on part of the arm until itclicks and locks into the catch. Because of leverage, the user need notapply great force. A force of 4 or 5 pounds is effective to push thecartridge down against the pneumatic boss sufficient to guarantee asound pneumatic interface seal.

Also on the subassembly are, not shown here, there are a number ofpneumatic channels that lead back to the manifold with valves located onit, carried by the X, Y stage. These are the ports for vacuum andpressure. Each of the actuation ports is controlled by a solenoid valvein the valve bank. It can switch each one of these ports to eithervacuum or pressure.

In the epi-fluorescent optical system is a laser diode, red laser diode,a collimator lens, a cylindrical lens, and three filters. An excitationfilter ensures any of the excitation light is within a certainwavelength band. There is a dichroic beam splitter which has a highreflectivity for the red of excitation 640 nanometers, but very lowreflectivity for the deeper red that comes back as a result of thefluorescence, around 680-690 nanometers. The reflects 640, but 680transmits. The 680 coming through hits another filter, the emissionfilter. This allows only a small band—it blocks all red, and it allows asmall band. Following this is a focusing lens onto a camera, called atube lens.

A cylindrical lens in the infinity space between the collimator and theinjector provides a stigmatic beam at the target. This produces a laserbeam at target of very long elliptical profile. The beam isapproximately 500 microns long by about 8 microns thick. It is like aline. The instrument scans that line down the channels to illuminate thewhole width of the channel.

Scanning an Array of Fixed Micro-Flow Elements

The novel arrays of elements described above are useful only ifeffectively read after the fluid assay is performed. The followingscanning apparatus, procedures and methods for automatically scanning amicrofluidic chip effectively solves the problem with arrays ofmicro-flow elements, and in particular, micro-length tube elements.

-   2 Scanner Description-   2.1 The Scanner-   2.2 The Scan-   2.3 Chip Layout-   2.4 Find Channels-   2.5 Find Elements-   2.6 Auto—Focus-   2.7 Auto—Exposure-   2.8 Fluorescence Scan-   2.9 Scan Data Processing    -   2.9.1 Load Data    -   2.9.2 Break data into segments    -   2.9.3 Thresholding    -   2.9.4 Locate Elements and Background in time history    -   2.9.5 Aggregate element mean RFU's into results        Introduction—Scanner

The general scanning concepts of invention are given in the blockdiagrams and flow charts. These are followed by description of aspecific, novel implementation. As a precursor to the specific method tobe described, the general capabilities of the scanner, a scan, and thegeneral microfluidic chip layout will be described first. These threesections are then followed by each sub-procedure in sequence thatcontributes to the overall method for automatically scanning amicrofluidic chip.

The Scanner

The scanner utilized in this method is a fixed, inverted epifluorescentmicroscope equipped with a three axis (x, y, and z) stage for motion ofthe chip to be read, a CCD camera for bright field imaging andfluorescence detection, a diode-pumped solid-state laser for excitation,and a white LED for bright field illumination. A cylindrical lens isused in the laser optical path prior to any filters to expand the beamsize. This allows the excitation of a larger surface area in a singlepass and allows for some flexibility when placing the elements in theflow channel during chip manufacturing. All of these scanner componentsare controllable via a computer as follows: on command x, y, z motion,image acquisition/imaging settings, laser on/off, and LED on/off. Themethod described herein uses various sequences and combinations of thescanner control/acquisition to orchestrate the automatic scan. SeeScanning for a general schematic of the scanner.

The Scan

A scan is comprised of a sequence of steps configured with start/end (x,y) positions, z (focus) position, velocity, and a segment number. Whenthe end position of a step in the sequence is not the start position ofthe next step, the stage will make a full speed move to the x, y, zposition of the start of the next step in the sequence, and no data iscollected during this rapid move. While executing one of the steps(moving from start to end in x, y at a fixed z) data is collected versustime. The data that is collected includes time, the step segment number(assigned while configuring a scan step), the present x and y positions,the camera settings (gain, exposure etc.), and information extractedfrom the images in the video stream from the camera. The informationextracted from the camera video is based on a region of interest (ROI).An ROI is defined as a rectangle somewhere in the image. The pixelswithin the ROI are processed to extract information from the image. Forexample the mean, median, standard deviation, max, min, etc. of thepixels inside the ROI from a given image are computed and included inthe data collected during a step move. At the conclusion of a scan, thedata collected throughout the sequence of steps that comprise the scanis written to a file (see sample in) that may then be processed toextract desired information.

The ability to flag various steps in a scan with a unique segment numberthat is subsequently written into the scan data file (“seeded into thefile”) greatly facilitates processing of scan data files andsignificantly improves the robustness of the various signal processingalgorithms. This capability is uniquely made use of a number of timesthroughout the auto-scan method.

All of the sub-procedures described in the subsequent sections are basedon the scan just described above, with the exception of the Auto-Focussub-procedure.

Chip Layout

A priori knowledge of the chip features/layout is necessary tofacilitate automatic scanning. A chip layout is depicted in Scanning. Inthis Figure, the chip itself is a 25×75 mm standard microscope glassslide. Within the chip is the scan zone that contains the elements ofinterest. The ‘scan zone—zoom’ provides more detail. In particular,Scanning shows that there are a number of fluidic channels (1 thru nleft to right in the Figure) and there are a number of elements per flowchannel (1 thru n top to bottom in the Figure). It is the elements thatare fluorescing are to be scanned in detail. The vertical lines in themiddle of the flow channels depict the scan moves that are executed forthe purposes of locating the channels, and the horizontal lines aboveand below the elements depict the scan moves that are executed for thepurposes of locating the elements and subsequently performing thefluorescence measurements.

Find Flow Channels

The first step in the automatic scan is to determine where the channelsare located relative to the stage x, y positions, and to also determinethe skew of the chip in the event that the channels are not exactlyparallel with the vertical axis. Predefined x, y positions based on apreviously complete homing of the scanner stage are sufficient forguaranteeing that the scan executed will, in fact, pass over all thechannel edges, given that the chip is mechanically referenced to thestage. The issue is then to determine precisely where the channel edgesare relative to the x, y stage positions. To this end, a ‘find channels’scan is executed following the horizontal lines in the middle of thechannels shown in Scanning. More specifically, the find channels scan isbroken into distinct steps such that there is a step across eachindividual channel at the ‘top’ and the ‘bottom’ of the scan zone thatis tagged with a unique segment number in order to facilitate subsequentdata processing. Further, the scan is done in bright field (i.e. laseroff, LED on), the ROI used is very narrow in width and extends the fullheight of the image (as show in Scanning), and the z position isintentionally ‘defocused’ from a nominal focused z position of zero, asestablished by homing the stage.

Alternatively, the ‘Find Channels’ routine can be done during the‘detect’ flow phase of the chip assay. In this mode, the channels arefilled with fluorescent dye. The scan to find the channels is then doneas described above but the scanner is in fluorescence mode (i.e. laseron, LED off). This has the advantage that the signal to noise ratio isvery high.

An example of the data collected during a ‘find channels’ scan is givenin Scanning and a zoom in to a single channel scan is given in Scanning.In this example the ‘find channels’ scan has eight steps, one for eachchannel crossing above and below the elements.

A scan data file from a find channels scan is processed on a per scansegment basis. Consequently, the data processing operates on a set ofdata as depicted. The data processing proceeds as shown in Scanning. Inthe Figure, the data processing will produce a scan configuration thatcan be used for the find micro-length tube elements procedure, or itwill throw an error that will halt the auto-scan procedure. Theinformation collected during this procedure is useful for:

-   -   Defining the Find Elements Scan.    -   Defining the Fluorescence Scan.    -   Collecting Chip Quality Control data about:    -   Variations in channel width    -   Variations in channel to chip reference edge        Find Micro-Length Tube Elements

The primary goal of find elements is to locate the first micro-lengthtube element in each flow channel. This information is then subsequentlyused for executing the auto-focus and auto-exposure procedures. The findchannels procedure must be a precursor to this procedure in that thescan utilized by find elements is built via knowledge of the channelpositions. The find elements scan is broken into a segment per channeland follows the horizontal lines as depicted in Scanning. This scan isdone in bright field (i.e. laser off, LED on), and the ROI used is widerthan a channel in width and has a height greater than the length of amicro-length tube element. This ROI is shown in Scanning. Further, thescan z position is intentionally ‘defocused’ from a nominal focused zposition of zero, as established by homing the stage.

An example of the data collected during a ‘find elements’ scan is givenin Scanning, and the processing sequence of the ‘find elements’ scandata is given as a flowchart in Scanning. The results of processing thisscan data are the x, y position of each element in each channel. Theinformation collected during this procedure is useful for: The x, ypositions to execute Auto-Focus.

-   -   The x, y positions to execute Auto-Exposure.    -   The x, y positions for processing a Fluorescence Scan.    -   Validating that the predetermined number of elements are in fact        present on the chip.    -   Collecting Chip Quality Control data about element placement.        Auto—Focus

This procedure takes as input the x, y positions of the firstmicro-length tube element in each channel as determined by the ‘findelements’ procedure. For each of these positions the procedure moves tothe given x, y position and conducts a sweep of z from a negativeposition thru zero to a positive position. While the z sweep is takingplace the full images from the camera video stream are run thru a Sobeledge detect filter and then the resulting image standard deviation iscomputed. The end result is a set of data as shown in Scanning. For eachsegment (i.e. at the x, y position of an element), the resulting zposition versus standard deviation plot is then used to find the zposition at the maximum value of the standard deviation (See Scanningfor details). The z positions, at the maximum standard deviation, fromeach segment are the ‘in focus’ z positions for each channel on thechip. The information collected during this procedure is useful for:Setting the focus for the Fluorescence Scan,

-   -   Gauging the degree to which the chip/and or stage is not flat        with respect to the optical system.        Auto—Exposure

The purpose of this procedure is to select the appropriate cameraexposure setting in order to efficiently utilize the range of the cameragiven the fluorescence level of the micro-length tube elements. Tooshort of an exposure will lead to dark images, poor signal to noise, andunderutilizes the camera range. Too long of an exposure will lead tosaturated images that cannot be used for collecting a fluorescencemeasurement. The degree to which a micro-length tube element fluorescesis dependent on the concentration of the targeted capture agent, e.g.antibody, in the sample under investigation and therefore will notnecessarily be known in advance. As a result, the best exposure settingmust be determined in-situ, for each fluid channel in the chip.

This procedure takes as input:

-   -   The x, y positions of the first micro-length tube element in        each channel as determined by the ‘find elements’ procedure.    -   Maximum pixel value range    -   Exposure range and start value    -   Scan Half Length    -   Data Extract Half Length

From this input, a scan, as depicted in Scanning, is constructed. Theauto-expose procedure then follows the sequence given in Scanning. TheROI used for this procedure is the same as that used for thefluorescence scan discussed in the next section. This procedure is donewith the LED off and the Laser on. To avoid significant photo-bleachingeffects the velocity of this scan is selected to minimize the laserexposure incurred by the element. The end result of this process is theoptimal exposure setting per channel to be used in the fluorescence scandiscussed next.

Fluorescence Scan

Using the (x, y) positions, z(focus) and camera exposure settings foundfrom the results of the ‘Channel Find’, ‘Element Find’, ‘Auto-Focus’,and ‘Auto-Exposure’ the Fluorescence Scan (FS) can be constructed. Aswith all measurements from the scanner, an ROI is used to collect pixelintensity values. The ROI for the fluorescence scan is a rectangleoriented with the long side perpendicular to the flow channel andpositioned in the image on top of the laser cross section (Scanning &28. The size of the ROI is determined by the size of the laser spot, thewidth of the fluorescent region on the micro-length tube element, thewidth of the channel containing the element, the number of pixels neededfor a measurement and the scan speed. These parameters can bepredetermined and optimized empirically and therefore do not change foreach FS. The FS starts at a location upstream of the first micro-lengthtube element in the channel to be scanned. The scanner is put intofluorescence mode (LED: Off, Laser: On) and the camera exposure time andz position (focus) are set. A scan is performed (see section 0).Scanning depicts a snapshot of an element during a FS.

Scan Data Processing

This sections discussion is available as a flowchart shown in Scanning.

Load Data

The data collected from the FS is loaded into memory and the mean ROIvalue is plotted vs. time (Scanning). This Figure depicts the mean ROIvalue for all the channels scanned vs. time.

Break Data into Segments

To process this data, it is broken up into segments where each segmentconsists of one flow channel's worth of data Q. A peak detectionalgorithm is used to determine the element positions in the channel withrespect to the background signal. The micro-length tube elementpositions found during the ‘Element Find’ can also be used to locate theelements in the segment.

Thresholding

Since the relative signal intensities for all micro-length tube elementsin one channel are approximately equal, k-means clustering can be usedto separate the pixels associated with the background from the pixelsassociated with the micro-length tube element. The outputs of theclustering algorithm are centroids representing the mean backgroundvalue and the mean element value. The mid-point between these twocentroids is used as a threshold. Thresholding must be done on achannel-by-channel basis due to differing background and exposuresettings per fluid channel.

Locate Elements and Background in Time History

Once a suitable threshold is found, the mean time history gets filteredusing a Savitzky-Golay (SG) filter and the peak detect algorithmidentifies all threshold crossings larger than a predetermined width,thereby rejecting of most of the high frequency noise in the data. Usingthe found threshold crossings the time history is further broken up intoan element signal component and a background signal component. Theelement signal component comes from the section of the channel with thefluorescent micro-length tube element in it. The background componentcomes from the ‘empty’ section of the channel adjacent to, butdownstream of the fluorescent element Q. This allows each micro-lengthtube element to have its own background-offset correction. The center ofeach component is found and the data points+/−25% of the element widthare then extracted to create an average value for each component (Seethe highlighted points in Scanning). Only points about the center of theelement and background are used in order to eliminate element edgeeffects. Since the signal rides on a background offset, the average ofthe background points is subtracted from the average of the elementpoints and the result is normalized for camera exposure and finallystored as that element's mean RFU (Relative Fluorescence Unit). This isperformed for each micro-length tube element in each channel on thechip.

Aggregate Element Mean RFU's into Results

Statistics are done on all micro-length tube elements' mean RFUs foreach channel and outliers are removed by finding the lowest % CV amongall combinations of element means. A minimum number of micro-length tubeelements must be retained for statistical purposes. These result valuescan now be applied to a standard dose curve to determine capture agent,e.g. antibody, concentration.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.

PDMS Membrane-Confined Nucleic Acid and Antibody/Antigen-FunctionalizedMicro-Length Tube Capture Elements, Systems Employing Them, and Methodsof Their Use”

Introduction

Assay devices constructed with functionalized micro-length tubes as havebeen described, and particularly glass nano-reactors (GNRs), haveimportant uses with respect to nucleic acid detection, to DNA assays andgenerally to the field of genomics detection (For uses see MastichiadisC, Niotis A E, Petrou P S, Kakabakos S E, Misiakos K. Capillary-basedimmunoassays, immunosensors and DNA sensors—steps towards integrationand multi-analysis. TrAC Trends in Analytical Chemistry 2008;27:771-84.))

Many of these uses employ fluorescent dyes that produce fluorescencewhen stimulated by excitation energy, in particular, by epi-fluorescentdetection, and especially when a laser is the excitation source.

Detection of Hybridized DNA:

While we refer to DNA probes immobilized on GNRs to detect DNA moleculesin solution, it is understood that this extends more broadly to nucleicacids in general, and can include RNA probes immobilized on GNRs tohybridize RNA molecules in solution, DNA probes to capture RNA insolution and RNA probes to capture solution-phase DNA molecules.

Implementations employ functionalizing a GNR with an appropriate DNAcapture agent (or probes) and spiking in a known concentration of DNAoligomers (or targets). Those DNA targets are then flowed with thesample (or other fluid of interest) through the system and hybridized tothe complementary strands immobilized on the DNA GNRs. At sufficientconcentrations and using suitable detection systems, these hybridizedmolecules can generate a detectable fluorescent signal indicating thatflow has occurred across the GNR, or an indication of the quantificationof that flow.

Detection Systems and Processes:

This hybridized DNA is typically a synthetic molecule custom synthesizedfrom any of the commercially available oligo supply houses (e.g.Integrated DNA Technologies IDTDNA.com), or an amplicon, the end-productof an amplification reaction such as PCR, or various isothermalreactions (Hyberbranched Amplification, Helicase reactions, qPCR,cold-PCR, etc), as these methods can easily generate the relatively highconcentrations of DNA required for fluorescent detection. These higherconcentration DNA hybridization events can be visualized either by theuse of an intercalating agent such as Sybrgreen (Life Technologies) orEthidium Bromide. Those dyes, when excited as by laser, generate asignal if they have become inserted between the strands of a DNA doublehelix, but generate no, or reduced, signal when present with only asingle strand. Alternatively, the DNA strands in solution can bedirectly fluorescently labeled either during commercial synthesis, orusing any of the chemical or enzymatic methods known to those practicedin the art (e.g. PCR with labeled nucleotides, etc). In this manner DNAstrands that are sufficiently complimentary to the “capture” strandsimmobilized on the GNR are bound to the GNR, and thus confer fluorescentsignal when excited by a laser.

In other cases, the DNA immobilized on the GNR is modified to generatesignal following hybridization. DNA strands known in the art as hairpinprobes (including so-called molecular beacons, scorpion probes and thelike) are specifically designed to fluoresce, or increase influorescence, following the hybridization of a complimentary molecule.Although there can be modifications, a typical design includes a DNAmolecule that possess a fluorescent dye and a quencher molecule locatedon different bases of the molecule, typically at the distal ends of thestrand. These probes are typically self-complementary; hence normallyfound in a closed, hairpin confirmation wherein the fluor and thequencher are in close proximity and non-fluorescent. When the probesbinds to a complimentary sequence, however, the molecule becomes linearand double stranded, separating the fluor from the quencher andfluorescence results.

We understand that it is also possible to generate detectable,quantifiable fluorescence resulting from lower concentration DNAhybridization events, such as those typically seen with populations ofnative, unamplified DNA, or levels seen circulating in the bloodstreameven without DNA amplification. In such cases the signal resulting froma hybridization event is amplified, as opposed to amplifying thehybridizing DNA itself. Examples of this include, but are not limitedto, the use of Quantum Dots, which have extremely high levels offluorescence per molecule, and other processes including Tyramide SignalAmplification (TSA) Systems (PerkinElmer), 3DNA Array Detection(Genisphere, LLC) and Rolling Circle Amplification that generatemultiple fluors per individual binding event.

Applications:

One application is in the context of a protein assay device, as in aportable cartridge constructed for protein assays. This useful mode ofutilization of DNA employs, in a single channel through which sample orother fluid of interest flows, one or more GNR's functionalized for oneor more protein assays, and one or more additional GNRs functionalizedfor capture detection of a given DNA strand. Here the capture anddetection of a DNA tracer is used as a run control, a confirmation ofappropriate fluid flow through the channel, and proper operation of thedevice (not for informational purpose from the sample). With a selectedfunctionalization for DNA, one achieves an orthogonal tracer that has noinfluence or cross-reactivity with ambient protein. This meets themonitoring need for situations in which adding or spiking-in anadditional protein to the sample fluid for monitoring purposes wouldproduce risk of cross-reaction or would otherwise be objectionable. Asimilar application utilizes a single sample flowing through multipleparallel channels, in which at least one channel is devoted to only oneor more protein assay GNRs, but another channel has one or more GNRsfunctionalized to DNA for monitoring purposes, that channel having onlyone or more GNRs functionalized for DNA or also including GNRs for otherpurposes, such as protein assay mentioned earlier.

This has direct application in the clinical research market as it givesclinicians a process to confirm whether the cartridge functioned asintended. It is simply a validation that the cartridge functionedproperly. This allows them to read negative results as likely a truenegative rather than a false negative resulting from an assay failure.

In other applications the DNA is employed as a signal calibrator, wherethe fluorescent signal from the DNA is compared to an expected signal,and the observed signal is either corrected to match the expected signal(the difference presumably due to differences in fluid flow rates, laserintensity or focus), or is simply confirmed to be within a predefinedrange, and hence acceptable, or outside the range, and thusunacceptable, thus alerting the user of possible poor quality data.

In other iterations, multiple types of GNRs are produced, eachcomplimentary to an individual population of DNA targets. These distinctGNRs can all be placed in the same channel, or can be spread acrossmultiple channels. When the various DNA target populations are spikedinto the sample at different concentrations, the data is used togenerate a calibration curve. This calibration curve is either used inconjunction with pre-determined relationships between DNA signal andprotein concentration to determine the concentration of proteinsdetected by antibody-coated GNRs, or can be used to calibrate the signalfrom one instrument with another, thus ensuring that data generated onone system is equivalent with that generated on another.

Capture of Native DNA from Samples:

GNR-based DNA capture also enables the detection or monitoring detect ofthe levels of a specific DNA sequence or sequences in a sample, whetherit be solely DNA detection, or a hybrid system in which protein and DNAare simultaneously detected using respective GNRs in a single system. Ineither case populations of GNRs are made with capture strandscomplementary to the sequence of interest, for instance a sequence incirculating blood or in cell lysate. As with the previous example of thecalibration curves, multiple different populations of GNRs, specific fordifferent DNA target populations can be generated and employed within asingle channel, or cartridge provided sufficient discrimination existsbetween hybridization conditions for the various target sequences. Insituations where a high concentration of DNA is available, DNAamplification is not required. The utility of non-amplified systemsdepends upon how many copies are present in the sample. For example whenone is looking at specific viral loads, depending on the level of avirus present in a patient, one may encounter suitably high levels ofDNA. Also in the case of transgenic organisms, multiple copies ofspecific genes introduced into the transgene are often present a highlevel.

For lower levels, amplification independent detection systems (TSA, RCA,3D-array) have been previously described. Alternatively, DNA targets caneither be amplified prior to analysis on the system the many processesknown to those versed in the art (PCR, etc) or a system of amplificationis provided to enable amplification within the cartridge channelsthemselves (described later under CARTRIDGE DESIGN CONSIDERATIONS).

Regardless of whether DNA amplification is required, this technology hasapplication for monitoring and/or quantifying populations of organismsbased on their genetics. For example, genetically modified organisms canbe monitored both by detecting the genes spliced into the organismthrough the engineering process itself, but also the presence ofproteins that the introduced genes cause the organism to express. Thisprovides value in several ways; one is in confirming the presence of agenetically-modified organism if one was interested in promoting ormonitoring that—for example in ensuring that seeds sent to farmers wheresuccessfully modified, confirming that harvested crops expressed thebeneficial traits that the engineering was intended to provide (e.g. theincreased nutritional value in golden rice) or in determining to whatextent the traits are expressed in the final product (i.e. the relativesuccess of the engineering process). Alternatively, the process can beemployed to ensure organic crops are free of engineered seed. This is ofparticular interest to the organic farming profession as a whole, andcertain geographic areas such as Europe. This invention addresses therequirement to test multiple samples and analytes (DNA or protein)simultaneously with a relatively small footprint and high ease of use.This type of system is employed at transportation hubs, ports of entryand other areas where the crops are concentrated after harvest (grainsilos, etc.).

Diagnostic applications include the ability to monitor diseaseprogression via the patient's biological response (protein production,e.g. cytokines) and the level of the infectious agent (by DNAsignature). For example, in the case of flu outbreaks, one cansimultaneously monitoring a patient's cytokine levels while quantifyingthe relative abundance of flu strains as indicated by GNR's specificdiagnostic DNA sequences for the major flu subtypes.

The DNA-based GNR system can also be used as an enrichment application,where capture probes are immobilized to the GNR, and a sample is flowedthrough the channel, with complimentary sequence binding to the GNRs.The capture can be employed for one of two purposes. Either the capturedmaterial can be released in a subsequent wash step, and recovered fordownstream manipulation of the enriched nucleic acid, such as next-gensequencing. Conversely, the capture process can be employed to REMOVEunwanted sequence from a sample, as a form of subtractive hybridization.Here a high concentration, possibly confounding DNA species can beremoved from the sample to enable downstream manipulation of theresidual nucleic acid.

Cartridge Design Considerations:

The ability to intermingle the DNA and antibody GNRs in the same channelor place them in discrete channels provides the ability to eithermaximize plexity (the number of distinct analytes measured in one assay)or to employ substantially different reagents for the differentdetection systems if required. For example, if DNA detection requiresbuffers and conditions that are not amenable to antibody baseddetection, separate channels can be employed, while if the two systemsutilize similar conditions the two assays can be combined, saving realestate and enabling more assays to be run per cartridge. DNA bindingrates and specificities can be controlled through buffer composition andthe presence of additives such as salt, DMSO, TMAC/TMAO and free Mg++ tomention a few. Individually addressable channels permit the use ofchannel-specific additives or hybridization buffers designed to ensuremaximal hybridization conditions for the DNA probes.

When assays are separated by channels, additional opportunities arisefor tailoring the specific reaction conditions in a channel by channelmanner. For example, localized heating of microfluidic chips is known tothe field employing strategies including, but not limited to, diodes,resistive heaters, flexible heating tape (e.g. kapton heaters), peltierheating blocks, resistance heating of etched indium tin oxide coatedslides, and IR light exposure. Hybridization temperatures of givennucleic acids are determined by a number of factors, mainly the sequencelength and base content (the relative amount of A,C,T and Gnucleotides). If the temperature is too far above the hybridizationtemperature, the complimentary strands will not anneal. If it is toolow, non-specific binding of non-complimentary strands can result.Thermal control is allows the hybridization temperature of a givenchannel to be tailored to the optimized temperature for the nucleic acidhybridizing in the channel.

Not only can the channel temperature be controlled, but the spatialchannel separation, possibly in conjunction with thermalisolation/insulation strategies including air gaps, insulating foam orrubber or peltier cooling pads, allow the temperature of a given channelto be shifted dramatically relative to the temperature of a neighboringchannel. This provides a variety of benefits. Reaction temperatures areeasily adjusted to ensure optimal DNA hybridization temperatures,providing optimal specificity for DNA capture probes in each individualchannel. Also, thermal control of the individual channel permitscombination of thermally stabile and thermally labile assays. Assaysthat require or benefit from elevated temperature (such as nucleic acidhybridizations) can be run concurrently with assays that require stabileor reduced temperatures such as protein assays by isolating thermalexchange from one channel to the next.

Benefits of GNRs for DNA-Based Detection

Physical characteristics of the GNR itself provide substantial benefitsfor nucleic acid-based applications relative to substrates typicallyused in the industry. One significant advantage GNRs provide to anyapplication (DNA or antibody based) is that they are easily manufacturedin large, low cost batches. This enables quality control testing of astatistically relevant portion of the manufactured lot, andperformance-based acceptance PRIOR to placement in cartridge. This isnot possible in platforms that rely on spotted capture antibodies or DNAprobes such as microarray. Spots can only be QC'd after the microarrayis completed, and the spotting process itself is a variable one, withspot diameter and spotted concentrations changing during themanufacturing run. After the run is completed, a small section of thecompleted microarrays are sampled, and the entire run is accepted orrejected based on those results, with high material and labor costsincurred when scrappage occurs.

An additional advantage is seen when GNRs are compared to the varioustypes of beads used in other commercial platforms. These bead basedplatforms typically employ either a relatively dense pack of nonporousbeads in kind of a column layer, relatively large, porous, agarose orsome sort of scaffolded bead comprised of cross linked polymers resemblea whiffle ball or a dandelion. While it is possible to achieve highconcentrations of immobilized material on these beads, problems arisewhen they are exposed to samples containing biological debris, such astissue chunks, blood clots, or cellular, or even contaminants from theeveryday world such as threads, lint, dust or particulate matter. Due totheir porous nature, bead-based platforms are susceptible to clogging.For example, the pore diameters in these porous beads are typicallydescribed in terms of angstroms, through which even soluble proteins canexhibit difficulty passing. Cellular debris and other particulatesquickly clog the bead pores and impede the flow of any reagent throughthe bead, inhibiting the assay process and increasing the backpressurein the system, sometimes with catastrophic results.

In comparison, the GNR is designed as a straight pipe with an internaldiameter of 75 microns, (750000 angstroms), roughly 10 times thediameter of a red blood cell. As a result, the GNR is extremely tolerantof particulate material, which readily passes through the GNR and outinto waste without impeding analyte diffusion to the capture probesimmobilized on the walls of the GNR.

It should also be noted that large particular matter is easily filteredthe inclusion of a membrane filter directly under sample wells. In thismanifestation, even particulates sufficiently large to block the 75micron GNR would be easily prevented from entering the system. This hassignificant value in that it enables the use of whole blood as a sample,preventing the passage of clots, etc. into the system microfluidics, andeliminating time consuming and laborious centrifugation steps prior toanalysis of clinical samples.

Lastly, the process through which DNA and antibodies are immobilized onthe GNRs results in capture moieties attached to the internal surfaces,but not the external surfaces of the GNR. This enables robotic or otherpincer-based mechanical systems to physically place GNRs in the channelsof the cartridge during the manufacturing process without concern fordamaging the immobilized capture molecules. The fact that theimmobilization surface is protected, physically shielded by the outsideof the GNR tube, protected from any sort of mechanical damage or insultwhether it is in pick and place or handling in any form.

Novel systems employ the PDMS-confined micro-length tube elements withnucleic acid, antibody or antigen capture agent (i.e., probe)immobilized on internal surfaces of the elements: Besides thosepreviously described in the many examples above, are the following:

(1) In a given microfluidic channel, a combination of (a) one or moremicro-length tubes are internally functionalized with nucleic acidcapture agent and (b) one or more micro-length tubes are internallyfunctionalized with capture agent for antibody-antigen binding. Theagents are selected, and present within the micro-length tubes insufficient number of each type element, with active agent in sufficientconcentrations, to enable the nucleic-acid functionalized elements todetect a complementary tracer and serve as an assay control or in amonitoring system for an antibody-antigen assay conducted by successiveback- and forth flows within the microfluidic channel. An examplearrangement is illustrated in FIGS. 104, 104A and 104B.

The reverse can also be usefully employed. That implementation uses atracer for antibody-antigen binding with respect to a nucleic acidassay. As well, a tracer of one nucleic acid can be employed withrespect to an assay for another nucleic acid, using appropriatelyfunctionalized micro-length tubes for capture of the tracer and for theassay.

Typically, in any of these cases, more micro-length tube elements areprovided for the assay than for tracer detection. In a preferredimplementation, the micro-length tubes of each functionalization arepre-formed en masse, as by dicing long drawn tubing, batchfunctionalized with respective capture agents, and micro-length tubeelements from the batch are located in the micro-fluidic channel. Thiscan be done by a pick-and-place instrument, such as tweezer or vacuumtip instrument, which may be manual or under automated control aspreviously described.

The microfluidic system may be provided in a portable cartridge, devotedto a single sample, or multiple microfluidic networks may be provided,having respectively different sample wells or sources.

The capture protocol is preferably implemented with flows of successivesample, wash and reagent(s), each flow phase including a succession ofback and forth movements of a given slug of a given fluid, slugdimension of the order of 100 times the length of a micro-length tubeelement, with sufficient number of successive slugs of that fluid tocarry out the intended phase of the assay, before the next fluid of theassay sequence is introduced.

(2) In a given microfluidic channel, a series of micro-length tubeelements immobilizing a nucleic acid capture agent to their interiorsurfaces are provided for passive (i.e. without amplification)analytical detection of a native nucleic acid (or more than one) in asample. An example is given in FIG. 105. In some cases, a tracer asdescribed in (1) is also included. Again, in preferred implementations,the micro-length tubular elements are pre-formed en masse, batchfunctionalized with respective capture agents, and elements from thebatch are located in the channel, e.g. by a pick-and-place instrument,which may be manual or under the automated control shown. Themicrofluidic system may be provided in a portable cartridge, devoted toa single sample, or multiple microfluidic networks may be provided,having respectively different sample sources;(3) Using a plurality of parallel, isolated micro-channels connected toreceive portions of the same sample, differing sets of internallyfunctionalized micro-length tube elements are provided, for conductingmultiple independent assays on the same sample, each as described for(2), with or without a tracer as described in (1), at least some of themicro-length tube elements being functionalized with nucleic acid. Anexample is shown in FIG. 106. In preferred implementations, two or moreparallel channels receive sample from the same source and discharge to acommon waste receptacle, as shown previously. In a particularlyimportant implementation of (3), multiple channels are provided withnucleic acid immobilized within micro-length tubes, the nucleic acidsbeing different species in the respectively different channels, andprovisions are made for applying different reaction conditions to therespectively different channels, for instance, different temperatureconditions. An example is shown in FIG. 107.(4) In an antibody-antigen assay device having multiple parallelchannels receiving portions of the same sample, one or more nucleic acidprobes may be incorporated in each channel to detect a tracer, accordingto feature (1) above, thus to obtain indication of proper operation ofeach channel, which may be conducting a different assay from the rest.An example is given in FIG. 108.

In FIGS. 104, 104A, 104B, 105, 106, 107 and 108, for purposes ofillustration, but by no means limiting, the nucleic acids illustrated inthe figures are shown as DNA (i.e. single strand DNA), but like examplescan be employed with other forms of nucleic acid, for instance singlestrand RNA or mRNA. Similarly, the immobilized capture agent forantibody-antigen binding are shown as an antibody, but antigens canalternatively be immobilized as capture agents against antibody targets.Micro-length tubes of various compositions can be used, for instancetransparent plastic with low fluorescence, but the presently preferredform is glass, forming glass nano-reactors (GNRs), and those are shownin the following examples. In any case the tubes are preferably sectionsof drawn form, with the smooth internal surface characteristics of thedrawing process, in which the material is progressively drawn from aheated ingot or progressively emerges through a stationary die.

Where employed for fluorescent assays, the substance is chosen to betransparent to the wavelengths of fluorescence passing outwardly, and inthe case of stimulated fluorescent emission, also to the wave length ofthe stimulating radiation passing inwardly. In other cases, in which themicro-length tubes are employed to capture a target for extraction forother forms of assay or processing, the micro-length tubes need not betransparent.

FIGS. 104, 104A and 104B illustrate a microfluidic channel that is partof a microfluidic network having micro-valves and micro-pistons, all aspreviously described herein, that produce flows in the channel. Themicrofluidic channel is of width W, for instance 180 micron. A series ofmicro-length tube elements, e.g. GNR's, are held immobilized in thechannel by a PDMS membrane that forms the top of the channel. Forinstance the GNR's may have an outside diameter O.D. of 125 micron,inside diameter I.D. of 75 micron, and length L. of 250 micron. (Thefurther examples can employ similar dimensions, for example). Otherregions of the same PDMS membrane form pneumatically deflectableportions of pneumatically actuated valves and pistons that produce theindicated channel flow in response to positive and negative pneumaticpressure applied to respective deflection chambers, controlled by anetwork of pneumatic channels connectible to a pneumatic controller, allas previously described.

In the series of GNRs in the micro-fluidic channel, the inner surface ofthe first two GNRs carry immobilized DNA for capturing a target tracerand the following four GNRs carry immobilized antibodies, e.g. forassay. This arrangement is useful in an antibody detection platform inwhich the DNA-immobilized GNRs are used as a control for the executionof the assay, i.e. to determine that the right fluid has flowed at theright rate and right duration. This arrangement is also useful topassively detect (i.e. without amplification) native DNA occurring athigh enough concentrations not requiring biological or signalamplification. In this particular case both DNA and antibody detectionoccur in a single channel using immobilized GN's.

In the case of use for control, a tracer is spiked into the samplecontaining a complementary nucleic acid strand, e.g., a DNA strand tobind to an immobilized DNA strand on the internal surface of the GNR.

For monitoring, a specific complementary strand of nucleic acid, e.g.RNA or DNA, is spiked into a fluid, for example one of the reagents usedfor the execution of a quantitative antibody test. At the end, presenceof the complementary strand is determined using fluorescent detection.The resultant signal detected from the nucleic acid immobilized GNRs iscompared to a defined acceptable range of values for the determinationof proper execution of the related step of the assay. Falling withinacceptance limits would be a positive indication that the respectivereagent was present and the protocol in this respect was executedproperly.

Another use with respect to nucleic acid, concerns immobilizing nucleicacid to the surface of the GNRs for capture of a native nucleic acid ina sample. Referring to FIG. 105, a series of 6 GNRs are placed in asingle channel, the internal surface of each GNR having immobilized DNAfor capture of native nucleic acid in a sample. The nucleic acid speciesmay be the same for the set of GNRs, e.g. for purpose of assayredundancy, or different, to detect different species. In this case itis preferable that the GNR-immobilizing PDMS membrane be permanentlybonded to structure forming the walls of the channel, to achieve arobust assay device. This may be by use of PDMS activated surfacebonding previously described herein, using the make and break techniqueat the associated micro-valves. An alternative use for the arrangementshown is to capture DNA for extraction and assay by other means or forfurther processing. In this case, the PDMS membrane is notsurface-activated during manufacture, and forms a removable bond with acooperating surface such as another layer of un-surface-activated PDMSin which channels sides are cut or glass forming the channel sides.

In FIG. 106, instead of having a single channel with immobilized nucleicacid GNR's, a series of channels is provided, illustrated here by 4parallel channels, in which a selected combination of nucleicacid-immobilized GNRs and antibody-immobilized GNRs are placed. Forexample, FIG. 106 illustrates channels 1 and 2 having nucleicacid-immobilized GNRs and channels 3 and 4 having antibody-immobilizedGNRs.

FIG. 107 again has 4 parallel channels with nucleic acid-immobilizedGNRs in channels 1 and 2 and antibody-immobilized GNRs in channels 3 and4, with the addition of selected channels being uniquely heated. In theexample, channel 1 is shown to be heated to a temperature of 50 degreesCelsius, channel 2 to a temperature of 37 degrees Celsius, and theremaining two channels heated to 32 degrees Celsius. The specificelevated heating of channels 1 and 2 are for the purpose of optimizingthe specificity of the nucleic acid binding properties for theparticular nucleic acids that are used in those channels.

FIG. 108 illustrates 4 channels with 3 GNRs in each channel, the first 2GNR's in each channel having antibodies immobilized on the GNRs whilethe 3^(rd) GNR in each channel has nucleic acid immobilized on the GNRfor the purpose of running control using the nucleic acid GNRs or, inthe alternative, for the purpose of passive detection of native DNA.

In all those cases involving assay detection of a captured target, oneof the many known techniques of detecting by fluorescence, andespecially stimulated fluorescent signal is employed, in many casesusing laser-excited epi-fluorescent scanning or imaging.

In other cases, as previously mentioned, it is desired to employ theunique system for harvesting the captured species or analyte for assayby other means or for further processing. In this case the PDMSmembrane, un-surface activated, serves the useful purpose of forming adetachable bond to a like surface of PDMS or other surface such asglass. After the capture or assay procedure is run, the membrane isremoved, preferably it being a bonded part to a pneumatically controllayer removed with it. This exposes for removal the GNRs carryinginternally the captured material, nucleic acid, antibody or antigen (orwith other functionalized elements, other captured entity). A usefulmeans of removing the elements is using tools the same or similar tothose used in placing the elements in the first place, whether handtools or automated tools. FIGS. 109, 109A and 110 illustrate use of thesame tools previously described with respect to placing the elements,FIGS. 109 and 109A illustrating removal of a micro-length element withtweezers in the case of channel width being greater than width of themicro-tubes, while FIG. 110 illustrate removing a GNR with tweezers fora channel in which the micro-length elements have been force-fit.

In general, a theory regarding ambient analyte optimization, isdescribed by Roger Ekin, in certain papers, including: R. P. Ekins,“Towards immunoassays of greater sensitivity, specificity, and speed: anoverview”, in: A. Albertini, R. Ekins (Eds.), “Monoclonal Antibodies andDevelopments in Immunoassay”, Elsevier/North-Holland, Biomedical,Amsterdam, 1981, pp. 3-21, which is incorporated herein by reference tothe extent necessary to understand the present invention, makes severalkey predictions, and is contingent upon choosing assay conditions suchthat the reaction kinetics proceed in the ambient analyte regime. Therequirements for, and various benefits of, operating in ambient analyte(“a.a.”) regime, include the following: There is superiorsignal-to-noise and limits of detection, which maximizes the detectionsignal relative to background noise; the assay results are not affectedby variations in sample volume, such as pipetting errors or othervariations; the assay results are not affected by variations of capturesurface area, typically based on surface area variations caused bymanufacturing of the GNRs; the ambient analyte (a.a.) condition isapproximated by the relationship: # of binding sites on the GNR<0.1 VKd(where V=Sample Volume, and Kd=the equilibrium dissociation constant);for a GNR having dimensions (in microns) of 265(L)×125(OD)×75(ID), theNumber of binding sites on the GNR is approximately 0.6 VKd, which isclose to (i.e., approaching) the desired ambient analyte condition butnot fully in that condition.

As is known, the ambient analyte (“a.a.”) theory is shown by the belowequation for f and optimization occurs when f (fractional occupancy ofbinding sites) is maximized in the below equation, where f is dependenton surface density of binding sites, surface area, reaction volume,equilibrium dissociation constant and the analyte concentration.

${f = \frac{\begin{matrix}{( {a + b + 1} ) -} \\\sqrt{( {a\; + \; B\; + \; 1} )^{2}\; - \;{4\;{ab}}}\end{matrix}}{2\; b}}{b = \frac{S\;\Gamma_{m}}{VKd}}{a = \frac{A_{0}}{Kd}}$f=fractional occupancy Γ/Γ_(m)S=Surface AreaΓ_(m)=max surface concentration of occupied receptors (moles/cm²)Γ=surface concentration of occupied receptors (moles/cm²)V=reaction volume (L)A₀=Analyte concentration (moles/L)Kd=Equilibrium dissociation constant (M)

Referring to FIGS. 111 and 112, the ambient analyte design condition isshown. In particular, users typically desire to use the least amount ofprecious sample possible. Also, 50 ul (microliters) was chosen as theminimum reaction volume required by a user to make multi-analytemeasurement. Ultra high affinity antibodies have Kd's around 10-50 pM(picomoles). It is desirable to use these in the a.a. region to realizethe full benefit of these highly sensitive antibodies. Also, highaffinity Ab's require less surface area to remain in the ambient analyteregion. For present configuration (3 GNRs, 250×75 um and 50 ul reagentvolume) the minimum Kd still in the a.a. region is 130 pM. Accordingly,reducing the GNR size to about 150×40 um would decrease the minimum Kdfrom 130 pM to about 60 pM. Also, increasing the minimum reagent volumeto 100 ul would decrease the minimum Kd to about 25 pM. Any or all ofthe above can be done to further improve assay performance and get evencloser to the ambient analyte condition. Other sample volumes can beused if desired, depending on the performance requirements of the assayin view of that discussed herein.

Referring to FIG. 113, the curve shows results of a surface areatitration assay, showing the fractional occupancy vs. surface area. Inparticular, for this experiment: Surface Area (SA) varied by populatingchannels with either 1, 2, 3, 4 or 5 GNRs. Present configuration uses 3GNRs (as seen on graph, not ambient analyte region). Total Surface Area(SA)˜2e5 sq. um (or 2e-3 sq. cm). This shows that for 1 GNR (withdimensions 250×75) OR 3 GNRs (with dimensions 150×25), and an OD of 125microns for both, the assay is essentially in the ambient analyte(“a.a.”) regime for Kd=60 pM. Estimated SA variance due to manufacturingprocesses 7e3 sq. um. Sensitivity to SA variation ˜2.5e-6%/sq. um.Anticipated % CV due to GNR surface area fluctuation is about <1%, whichis a desirable result. Even though the assay in this example is notoperating in the full ambient analyte region, the GNR surface area istightly controlled enough in the GNR manufacturing processes tosufficiently reduce signal variation due to GNR surface area variation.Thus, decreasing the surface area of the GNRs from 250×125×75 to asmaller surface area, would further reduce sensitivity to surface areavariation and optimize performance with even high affinity Ab's(antibodies), as discussed herein.

An estimate of Antibody surface density for the GNR is as shown in thebelow table:

Ab dia 8 nm Ab diam 0.008 um area 5.02655E−05 um{circumflex over ( )}2density 19,894 per um{circumflex over ( )}2 % of sites active 20% activedensity 3,979 per um{circumflex over ( )}2 mass 15,000 Da density6.6E−21 moles/um{circumflex over ( )}2 density 6.6E−13moles/cm{circumflex over ( )}2 Surface Density 3979 Sites/um{circumflexover ( )}2

Referring to FIG. 114, the graph shows the dose curve dependence onreaction volume. In particular, a rapid decrease in curve sensitivityoccurs for reaction volumes less than 20 ul. Also, curve shape remainsnearly unaffected for reaction volumes greater than 40 ul. Therefore, aminimum reaction volume of about 40 ul is desired to perform the assayand meet the desired performance criteria discussed herein. Other samplevolumes may be used; however, the performance may be degraded. The modelparameters for graph shown are: Kd=60 pM; Surface Density=3,000sites/SQ. um; and Surface Area (SA)=0.002 sq. cm for 3 GNRs, at 250×75um.

Referring to FIG. 115, illustrations (a)-(d), for the dose curves shown,the assays appears to be substantially insensitive to consumed samplevolume. Accordingly, the GNR capture surface has full access to reactionvolume and sample homogenization (or mixing or equilibrating) isoccurring as a result of piston reciprocation.

Referring to FIG. 116, in performing a reaction volume titrationexperiment, we found the following: decreasing reaction volumes resultin decreasing signal, especially below 40 ul; the signal becomes almostindependent of reaction volume above 50 ul; in this experiment with 3GNRs (250×75) and 4 analytes, the consumed volume less than 5 ul, andvaried volume of sample in reservoir from 15 to 50 ul. Also, from amathematical model, Kd=60 pM, 3000 capture sites/sq. um, for 3 GNRs(250×75 um).

In view of the analysis shown herein regarding ambient analyte, we havefound that to further optimize approaching the ambient analyte conditionthe GNRs may be made on the order of about 150 microns long, about 125microns outer diameter (or possibly about 100 microns), and about 25microns inner diameter. Such dimensions would be achievable with currentmanufacturing limitations (drawing, dicing). This also takes intoaccount potential end face effects of about 20% of the length and alimiting the inner diameter to a minimum of about 25 microns to avoidpotential clogging issues. In that case the Surface Area (SA) is reducedfrom about 0.02 sq. cm (with a GNR of 250(L)×125(OD)×75(ID) microns) toabout 0.0007 sq. cm (with a GNR of 150(L)×125(OD)×25(ID) microns). Otherdimensions may be used but may not optimize these parameters.

FIGS. 117 and 118, shows the fluidic architecture for some of theanalysis performed herein, which includes: 8 circuit fluidic layer (2fluidic layers per cartridge); Circuits are fluidically isolated fromone another; Each circuit contains 4 isolation channels with 3 GNRseach; and Each isolation channel is used to measure a unique analyte;and the piston volume is 0.3 microliters.

FIGS. 119 and 120, show the results of a mixing experiment where:Circuit Volume=2.1 ul; Fluidic circuit initially filled with fluorescentdye; Sample reservoir initially filled with clear buffer (50 ul); Pistonactuation: 100 ms open state followed by 100 ms closed state; Continuouspiston actuation for 60 seconds; Fluorescence interrogated near GNRlocation.

The observations from this experiment were as follows: Peak signal foreach piston stroke decays over time, indicating that the buffer and thedye are mixing over time; Final fluorescence signal is asymptoticallyreached at about 40 seconds, indicating that buffer and dye arecompletely mixed in 40 sec.; Final signal level is predicted by scalinginitial signal by dilution factor; DF (Dilution Factor)=(circuitvolume+sample volume)/circuit volume. Accordingly, we have shown thatfor at least one embodiment of the system of the present invention,complete mixing of the sample volume occurs.

In one embodiment, reagent reciprocation (also referred to herein aspiston sloshing, includes the following parameters: Pulse volume=1.2 ul(4×0.3 ul); Pulse Duration=50 ms; AVG volumetric Flow Rate=24 ul/s;Slosh Period=200 ms (open to close states); piston volume=0.3microliters.

Accordingly, reagent reciprocation provides full access to the bulksample volume and eliminates sample diffusion limitations near thesurface of capture surface of the GNRs. More specifically, we have foundthat in a stationary condition, where a portion of the sample volume isbrought into the reaction channel and allowed to sit undisturbed and incontact with the capture surface for a period of time, the analyteconcentration would become depleted as a result of rapid binding to thecapture surface. This is exacerbated in microfluidic channels due totheir very large length to width aspect ratios. Since diffusiondistances may realistically only be about 3 mm over the span of a 40 minincubation period and the fact that the channel volume is very low,typically about 1.0 ul, only a small portion of the reaction volumewould interact with the GNR capture surface. Rapid reciprocation servesto replenish the concentration locally by carrying the depleted sampleback to the sample reservoir where it is homogenized (or equilibrated)with the full reaction volume, thereby removing diffusion limitations onthe reaction kinetics and thus maximizing the utilization of the samplereaction volume.

The micro-length tube element 32 may also be referred to herein as a“GNR” (glass nano reactor), a species of micro-length tube, or as“micro-flow element”, “microtube”, or “micro-bore tube”, all intended torefer to a tubular element of micro length; a micro-length tube in thecontext of a detection element captured in a channel, which can beregarded as an unique and advantageous species of a “micro element”, a“microparticle” or a “micro-length particle”.

As referred to herein, a “batch” (or bulk number) of the GNRs 32, istypically the amount of GNRs located in the Eppendorff tube (or othertube capable of performing the functions described herein) that isexposed to the capture agent, and is at least thousands, e.g., typicallyabout 75,000 to 150,000. In that case, each tube of GNRs 32 constitutesa “batch.”

Regarding removal of capture agent from the exterior of the GNR 32 (asdiscussed herein), it is known for typical particle assays to use gentleagitation of the particles (in this case the GNRs) during silanizationand functionalization, e.g., using magnetic stir bars, a rotisserie,gentle rocking, gentle vortexing, or other techniques to move the GNRsaround in the fluid (or fluid around the GNRs), to achieve good mixing.However, we found that when gentle vortexing was done during GNRfunctionalization, some of the capture agent was removed from the outersurface of the GNR 32. This caused further experimentation, whichresulted in the discovery that all of the capture agent can be removedfrom the exterior cylindrical surface of the GNRs 32 by vigorousvortexing as described herein.

The appropriate vortexing speed and diameter, is dependent on the natureof the suspension liquid, e.g., the viscosity of the liquid chosen, andsize of the GNRs 32, and can be easily determined experimentally. It isset by observing whether the capture agent is effectively non-existenton the outside, long surface of the GNRs 32 (e.g., the outsidecylindrical surface in the case of the body being of circularcross-section), while also ensuring that the internal coating along theaxial length of the GNR 32 is substantially uniform (as discussedherein).

After the GNRs 32 are allowed to roam on the alignment plate 70, theyfall into the alignment pockets still in the presence of the stabilizingsolution. The alignment plate 70 may be gently rocked or agitated, tofacilitate the GNR capture process. For example, the plate 70 may befirst rocked about the longitudinal axis of the pockets allowing many ofthe GNRs to fall into the pockets, then rocked about an axisperpendicular (or other orientation) to the axis of the pockets toredistribute the remaining GNRs, then switch back to the longitudinalaxis, and repeat until the GNRs populate most (e.g., more than 95%) ofthe pockets on the plate 70.

We have found that an accurate and repeatable biochemical assay withhighly sensitive quantitative results can be achieved without removingthe capture agent from the end faces of the GNR 32 (e.g., withoutablating the ends faces or otherwise removing capture agents from theend faces). In particular, this occurs because the surface area of theend faces are not significant enough to cause depletion problems, and,thus, can maintain a condition that substantially approaches or nearsthe ambient analyte theory conditions, and because the optical signalfrom the end faces are ignored (or filtered out) by the optical reader(as discussed hereinafter), and, thus, it does not contribute in anappreciable way to the optical noise floor.

Referring to FIG. 121, illustrations (a)-(d), regarding the length L, wehave found that the shorter the length L of the GNR 32, the greater theuniformity of the coating density of capture agent molecules along theinternal length L of the GNR 32 when coated by immersion as describedherein. In particular, when the length L of the GNR 32 is on the orderof about 1 mm or 1,000 microns (illustration (a)), an axial coatingdensity curve (or profile) 610 showing the capture agent moleculecoating density along the internal axial (or longitudinal) length of theGNR 32, shows a significant portion of the axial length (near the middleof the GNR length) with very low coating density, which is verydetrimental to obtaining sufficient binding to detect reagents and thusto obtaining sufficient optical signal for assay measurementreadings/results. When the GNR 32 has a length of about 750 microns, theaxial coating density profile 610 also shows a low coating density nearthe middle of the GNR length, but not as severe as the 1 mm length(illustration (b)).

We have found that as the length L of the GNRs 32 gets shorter thanabout 750 microns, the uniformity of the capture agent coating densitycurve 610 of the inner surface of the GNR 32 improves significantly. Inparticular, the coating density curve 610 becomes more uniform when thelength L is about 500 microns, but still exhibits a decrease in coatingdensity (illustration (c)) near the middle of the GNR length. When thelength L is about 250 microns or less (illustration (d)), the coatingdensity curve 610 is substantially constant along the entire length L ofthe GNR 32, thereby providing maximum binding to detect reagents and,thus, maximum optical signal for the assay measurement results.

We have also found that even if the axial coating density curve 610 fora given GNR length L is not completely uniform (or flat) over the entireGNR length, it will not adversely affect the performance or quality ofthe assay results, provided the coating density profile is consistentfrom one GNR to the next (for a given GNR batch) and any non-uniformitydoes not have any substantial negative effects on the opticalmeasurement of the assay results.

In addition to the axial coating density decreasing significantly forlonger GNR lengths, there are also optical edge effects caused bycapture agent on the two end faces of the GNR and possibly other opticaleffects, such as reflection or refraction of incident light from theedge faces. Such effects can distort the optical measurement signal forthe assay results. As a result of these edge effects, a certain distancefrom the end of the GNR 32 (e.g., about 15 microns) may not be usablefor assay measurement purposes, as indicated by the vertical dashedlines 612. Accordingly, the usable region of flat or uniform coatedinner surface of the GNR 32 is further reduced by the edge effects 612.

In addition, another advantage of shorter GNRs is that they are moreamenable to withstanding axial tweezing forces during pick and placemotions.

Referring to FIGS. 122 and 123, an assay cartridge creation process anda process for running an assay with the present invention is shown.

Referring to FIG. 124, a top view of the fluidic and pneumatic channelsare shown for a cartridge having 16 sample wells, 16 fluidic circuits(each having 4 channels with GNRs) and 64 detection analyte input ports.It also shows how the fluidic and pneumatic channels intersect with thevalve and piston features of the present invention. Referring to FIG.125, a top view of the pneumatic channels laser cut (or otherwisemachined or formed) on the underside of the reservoir member is shownfor the channels. More specifically, the pneumatic channels providepaths for actuating the internal valves and pistons for moving fluids,as described herein. The pneumatic channels are closed by a double-sidedadhesive sheet (PSA layer), pressure sensitive adhesive layer, whichlies between the PDMS membrane and the reservoir layer. The PSA layerhas cut-outs (or through holes of various geometries), that definepneumatic cavities for the valves and pistons, into which the membranedeflects in response to pneumatic negative pressure (or vacuum) appliedby the pneumatic channels which are cut into the reservoir layer, asdescribed hereinabove.

As discussed herein, bypass flow is provided around the GNRs 32 of thepresent multi-analyte detection system, in which multiple channelsdischarge sample fluid back to the sample chamber for mixing. Suchbypass flow has several benefits. In particular, it enables full samplemixing, thereby allowing the entire sample volume to be used in theassay, which allows the sample volume to be substantially independent ofthe number of analyte channels, and allows the system to be sized forminimum sample volume. In addition, it decouples the overall channelflow impedance from the GNR internal flow impedance, thereby enablingboth flow effects to be optimized. In addition, having such bypass flowdoes not impact the repeatability and consistency of the measurement.

In particular, the flow mixing of the present invention provides asingle source volume (e.g., 50 microliters) of a multi-analyte sample inthe well, which can be used with N separate and different analyte testchannels (or branches), where the sample volume is substantiallyindependent of the number of analyte channels, provided the samplevolume is a least as large as the total dead volume of the fluidicscircuit. This is because of mixing of the entire sample volume thatoccurs between sample flowing in the channels with GNRs and the samplewell, through the reciprocating flow, which is caused by repeatedactuation of the dedicated piston in each branch of the multi-analyteassay.

Regarding flow impedance, the local flow impedance around the GNR 32 inthe channels 30 is has two portions with very different impedances: i)the narrow internal detection passage along the inside of the GNR 32which has a high flow impedance; and ii) the by-pass flow outside theGNR 32 along a non-analyte-capturing path which has a generally lowerflow impedance.

This arrangement creates an overall low impedance to flow through thebranch, which enables the sample liquid to flow at a relatively highvelocity (highest velocity being near the center of the channel 30) whenit is flowing back toward the sample well. This high velocity flowenables high efficiency mixing with the sample well contents upon eachreciprocation of the piston. As a result of such mixing, the sampleliquid in each trip through the particular branch contains a refreshedconcentration of the particular analyte molecules, such concentrationbeing of the order of the original sample concentration, therebycreating a condition that approaches the ambient analyte detectionconditions. Accordingly, it is desirable for the channel flow impedanceto be low to allow such high velocity flow to occur easier.

The high impedance portion of the flow that passes through the inside ofthe GNR 32 and past the immobilized capture agent, enables molecularcapture to occur under quiescent flow conditions. This enables a highattachment efficiency of the analyte molecules and also avoidsdisturbing the molecular binding activity. In general, the internaldiameter is desired to be small to reduce the assay surface area toachieve the desired assay performance, as discussed herein. As a result,the internal GNR flow impedance is relatively high to allow optimizethese conditions.

Thus, this arrangement decouples the internal GNR flow impedance (whichis relatively high), from the overall channel flow impedance (which isdesired to be small), thereby allowing the design parameters of both tobe optimized for their respective functions.

According to the present invention, in a multiplex microfluidic assaydevice for determining concentration of multiple molecular analytes in asample fluid, for use with an assay system that includes microfluidicpump and valves, respective actuating controller for pumps and valves,and an assay reader, the assay device comprises: a sample well forreceiving sample fluid to be analyzed, a plurality of fluidicallyparallel detection channels, each defining a microfluidic volumeconnectable by a manifold to the sample well by an associated inletpath, and each detection channel containing an immobilized capture agentspecific to at least one of the plurality of analytes, the volume ofeach detection channel and its associated inlet path being a minorportion of the combined volume of the well, inlet path and, a pluralityof secondary reagent channels connectible to respective detectionchannels to introduce respective secondary reagents specific to theassays to be performed in the respective detection channels, thedetection channels being isolated or isolatable from one another duringpresence of respective secondary reagents to avoid cross-reactions, eachof the plurality of detection channels associated with a dedicatedreciprocal microfluidic pump operable by the operating system inrepetitive cycles, each cycle having an intake stroke that defines thevolume of a fluid segment and a discharge stroke, the pumps eachconstructed, during an analyte capture stage of the assay, in its intakestroke to move a microfluidic segment of sample from the common samplewell through the respective detection channel for exposure to theimmobilized capture agent, and in a discharge stroke move the samplesegment backwardly past the immobilized capture agent toward the inletpassage for further exposure to the immobilized capture agent, thedevice constructed to determine concentration of the respective analytesin the sample on the basis of capture of analytes substantiallydependent on the concentration value of analyte in the sample, whereineach reciprocal pump has intake stroke volume and velocity such thatduring each discharge stroke of the pump, at least a portion of thepreviously withdrawn fluid is returned beyond the respective detectionchannel for mixing with segments from other channels that are likewisereturned, such that during the analyte capture stage of the assay theconcentration of analyte in repeated segments of fluid sample passingthrough a given detection channel is not substantially depleted of therespective analyte due to previous sample capture cycles performed onsegments of liquid in that microfluidic channel.

According further to the invention, a multiplex assay device fordetermining concentrations of multiple analytes in a sample, the devicehaving multiplicity of microfluidic channels containing capture agentfor respectively different analytes, each channel fed a portion of thefluid sample from a common reservoir to expose the portions of thesample to the respective capture agents, the device constructed, foreach channel, following exposure of the flow to the respective captureagents, to direct at least a portion of the respective flows to returnto a common volume in which it is mixed with flows from the otherchannels, thus to provide a supply of substantially undepletedconcentration of the analytes for repetitive flows through the channels,the device constructed to determine concentration of the respectiveanalytes in the sample on the basis of capture of the respectiveanalytes in quantity substantially dependent on the concentration valueof the analyte in the original sample.

According further to the invention, wherein the common volume is atleast partly defined by a common manifold passage, and/or wherein thecommon volume is defined at least in part by the common reservoir. Stillfurther according to the present invention, wherein the flow paths forthe return flows comprise reverse direction flow in conduits originallyintroducing sample to the respective channels containing the captureagents.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.

What is claimed is:
 1. In assembling a portion of a microfluidic deviceby conducting bonding action by contacting faces of opposed bondablematerials, one of the bondable materials comprising a flexible sheet,the method of employing repeated make-and-break-contact manufacturingprotocol on a first region R1 of the contacting faces of the bondablematerials while maintaining continual contact of the faces in a secondregion R2 of the contacting faces until bonding is completed, therebyover time neutralizing the tendency for permanent bonds to form in thefirst region R1, thus enabling making and breaking actuated movements ofthe first region R1 of the flexible sheet relative to the portion of theother material that it opposes, and providing a chamber defining adeflection cavity into which the first region R1 of the flexible sheetdeflects during the make-and-break-contact manufacturing protocol. 2.The method of claim 1 in which the faces of opposed bondable materialshave the capability of forming molecular bonds, and molecular bondsbetween the faces are formed in the second region R2 of the continualcontact.
 3. The method of claim 2 in which the materials have thecapability of forming covalent bonds and covalent bonds between thefaces are formed in the second region R2 of the continual contact. 4.The method of claim 1 in which at least one of the materials is asurface-activated bondable material.
 5. The method of claim 4 in whichthe at least one of the materials is surface-activated PDMS.
 6. Themethod of claim 4 in which both materials are PDMS.
 7. The method ofclaim 4 in which the flexible sheet portion in the region R1 defines avalve diaphragm and the opposed portion of the other material defines avalve seat which the flexible sheet portion engages.
 8. The method ofclaim 4 in which the bonding of the flexible sheet material to theopposed material acts to close and seal a flow channel, fix the positionof an inserted element in a flow channel, or form a piston diaphragm ofa piston of a membrane pump.
 9. The method of claim 8 in which all theactions are simultaneously performed.
 10. The method of claim 1 in whichthe repeated make- and break-contact manufacturing protocol is performedby applying, respectively, positive and negative air pressure to theback of the region R1 portion of the flexible sheet via the deflectioncavity.
 11. The method of claim 10 in which the negative air pressure isof larger magnitude than the positive air pressure.
 12. The method ofclaim 4, comprising: bringing the region R2 of the faces of the twomaterials into continuing contact for bonding and applying negativepressure to the deflection cavity to draw the flexible sheet portion inthe region R1 into the deflection cavity, away from the oppositematerial; and thereafter, in cyclical manner, repeatedly applying apositive pressure pulse followed by a negative pressure pulse to thedeflection cavity to cause the flexible sheet portion in the region R1to repeatedly make and break contact with a surface portion of theopposite material during cure of the bond between the adjacent surfaceportions in the region R2 of the two materials that are in continuingcontact.
 13. The method of claim 1 further comprising applying anegative pressure to the deflection cavity prior to and during initialcontacting of the region R2 of the faces of the two materials forbonding, so that the flexible sheet portion in the region R1 does notbond at the time of initial contact of the region R2 of the faces of thematerials.
 14. The method of claim 1 in which the material comprisingthe flexible sheet has its face comprised of PDMS, the face having beensurface-activated.
 15. The method of claim 4 in which the material is alayer comprising PDMS throughout its thickness.
 16. The method of claim1 in which the flexible sheet is comprised substantially of a materialother than PDMS, but has its face opposing the other material comprisedof an activatable adhesive coating that has been surface-activated forbonding.
 17. The method of claim 16 in which the activatable adhesive isPDMS.
 18. The method of claim 14 in which the material opposite theflexible sheet has its face for bonding comprised of PDMS, the facebeing surface-activated.
 19. The method of claim 14 in which thematerial opposite the flexible sheet has its face for bonding comprisedof glass, presenting OH groups to the activated PDMS surface.
 20. Themethod of claim 14 in which the material opposite the flexible sheet hasa main body formed of a material, on the face of which is a linker layerbonded to that material, which presents OH groups to the activated PDMSsurface.
 21. The method of claim 20, wherein the material of the mainbody opposite the flexible sheet comprises synthetic resin.
 22. Themethod of claim 1, wherein the material opposite the first region R1 ofthe flexible sheet does not deflect during the bonding process.
 23. Themethod of claim 1, wherein the flexible sheet portion in the region R1is an elastomer subject to thinning under tension, and the flexiblesheet portion in the region R1 is deflected initially with a negativepressure of a first magnitude, and later with a negative pressure at asecond magnitude of larger value, the first magnitude assuring bondingof contacting faces in the region R2 up to the perimeter of the flexiblesheet portion in the region R1.
 24. The method of claim 14 in which thematerial opposite the flexible sheet has its face for bonding comprisedof a material presenting OH groups to the activated PDMS surface. 25.The method of claim 1, wherein the deflection chamber comprises an openslot defined by walls that engage the back side of the flexible sheet.26. The method of claim 1, wherein the deflection chamber compriseswalls and a ceiling, the ceiling creating a physical limit fordeflection of the flexible sheet into the chamber.
 27. The method ofclaim 1, further comprising applying positive and negative pressuresbetween the faces of the opposed materials in the region R1 to performthe make-and-break protocol.
 28. The method of claim 4 in which theflexible sheet portion in the region R1 defines a piston diaphragm of amembrane pump which deflects toward and away from the opposed portion ofthe other material.
 29. The method of claim 1, wherein there are aplurality of regions R1 and the make-and-break protocol is performedsimultaneously on the plurality of regions R1.
 30. The method of claim1, wherein the material opposite the flexible sheet in the region R1having a first flexible sheet portion, is also a flexible sheet having asecond flexible sheet portion opposite the first flexible sheet portion,and further comprising: providing, at the back of the second flexiblesheet, a second chamber defining a second deflection cavity into whichthe second flexible sheet portion may be deflected, away from contactwith the opposite material, while other regions of the faces of the twomaterials are in continual contact for bonding; bringing the faces ofthe two materials into continuing contact for bonding and applyingnegative pressure to the deflection cavities to draw each of theflexible sheet portions into its respective cavity, away from theopposite material; thereafter, in cyclical manner, repeatedly, insynchronized manner, applying a positive pressure pulse followed by anegative pressure pulse to each of the cavities to cause the opposingfaces of the first and second sheet portions to repeatedly make andbreak contact with the each other during cure of the bond between theadjacent face portions of the two materials that are in continualcontact.
 31. A method of making a microfluidic device, comprising:contacting faces of opposed bondable materials, at least one of thebondable materials comprising a flexible sheet; performing repeatedmake-and-break-contact protocol on a first region R1 of the contactingfaces of the bondable materials until the tendency for permanent bondsto form in the first region R1 has been neutralized; maintainingcontinual contact of the faces in a second region R2 of the contactingfaces until bonding is completed in the second region R2; providing achamber defining a deflection cavity into which the first region R1 ofthe flexible sheet is drawn during the make-and-break-contact protocol;and thereby enabling actuated movements of the first region R1 of theflexible sheet relative to the portion of the other material that itopposes.
 32. The method of claim 31, wherein the opposed bondablematerials comprises the flexible sheet and a second flexible sheet; andfurther comprising providing a second chamber defining a seconddeflection cavity into which the first region R1 of the second flexiblesheet is drawn away from contact with the opposite material during themake-and-break-contact protocol, thereby enabling actuated movements ofthe first region R1 of the flexible sheet and the second flexible sheetrelative to each other.
 33. A method of making a microfluidic devicehaving a microfluidic valve, comprising: contacting faces of opposedbondable materials, one of the bondable materials comprising a flexiblesheet, the faces comprising surface-activated PDMS; performing repeatedmake-and-break-contact protocol on a first region R1 of the contactingfaces of the bondable materials until the tendency for permanent bondsto form in the first region R1 has been neutralized; maintainingcontinual contact of the faces in a second region R2 of the contactingfaces at least until bonding is completed in the second region R2;providing a chamber defining a deflection cavity into which the firstregion R1 of the flexible sheet is drawn during themake-and-break-contact protocol; wherein the first region R1 of theflexible sheet portion defines a valve diaphragm of the microfluidicvalve and the opposed portion of the other material it opposes defines avalve seat of the microfluidic valve which the flexible sheet portionengages; and thereby enabling the valve diaphragm to perform actuatedmovements to engage and disengage with the valve seat, without thediaphragm bonding to the valve seat.
 34. The method of claim 33, whereinthe device comprises a plurality of valves and themake-and-break-contact protocol is performed simultaneously on theplurality of valves.